Creation and Improvement of a Yeast RNR3-lacZ Genotoxicity Testing System
A thesis Submitted to the College of Graduate Studies and Research in Partial Fulfillment of the Requirements for the Degree of Master of Science in Toxicology Graduate Program University of Saskatchewan
Xuming Jia, MD, M.Sc
©Copyright Xuming Jia, January 2003. All rights reserved.
+02..001 5410Y3 PERMISSION TO USE
In presenting this thesis in partial fulfillment of the requirements for a Master degree from the University of Saskatchewan, I agree that the libraries of this University may make it freely available for inspection. I further agree that permission for copying of this thesis in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my thesis work or, in their absence, by the
Head of the Department or the Dean of the College in which my thesis work was done. It is understood that any copying or publication or use of this thesis or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my thesis.
Requests for permission to copy or make other use of material in this thesis in whole or in part should be addressed to:
Chair of the Toxicology Group
Toxicology Centre
University of Saskatchewan
44 Campus Drive
Saskatoon SK
S7N 5B3 Canada ABSTRACT
A variety of environmental toxicants can damage DNA and thereby produce congenital malformation and cancer. In order to evaluate the genotoxic effects of environmental agents, numerous genotoxicity testing systems have been developed.
These tests either directly assess the genetic alterations (e.g., the Ames test) or indirectly measure the cellular response to DNA damage (e.g., SOS Chromotest)
With the knowledge obtained from studying molecular mechanisms of DNA damage and signal transduction in Saccharomyces cerevisiae, a sensitive and stable genotoxicity testing system was developed based on the induction of a S. cerevisiae
RNR3-lacZ reporter gene expression in response to a broad range of DNA-damaging agents and agents that interfere with DNA synthesis. The tested agents include kno\Vn carcinogenic and genotoxic agents, ranging from DNA alkylating agents, oxidative chemicals to ionizing radiation as well as some known non-genotoxic agents. All the tested known genotoxic agents were able to induce RNR3-lacZ expression at a sub-lethal dose. In particular. a potent colon carcinogen~ 1~ 2-dimethyl hydrazine, was not detected as a mutagen by a standard Ames test, but was able to induce RNR3-lacZ expression. In contrast. both non-mutagenic and non-genotoxic chemicals tested were unable to induce
RNR3-/acZ expression. The sensitivity and inducibility of three well-characterized yeast
DNA damage-inducible genes have been compared and it was found that RNR3 is more sensitive than RNR2 and MAG 1. The effects of agent dose, post-treatment incubation time and cell growth stage on RNR3-lacZ expression were also determined and optimized. In order to create a stable and user-friendly testing system, the RNR3-lacZ
11 cassette was integrated into the yeast genome to demonstrate that its inducibility is
indistinguishable from that of the plasmid-based studies.
Although the sensitivity of RNR3-lacZ testing is comparable to that of the Ames
test and SOS Chromotest, it was reasoned that the sensitivity might be further improved
by using yeast strains defective in certain DNA repair pathways. Hence, several deletion mutant strains from different repair pathways were used as host strains for the RNR3-lacZ test. It was found that the magi null mutation specifically enhanced the sensitivity of
RNR3-lacZ test in response to DNA alkylating agents such as methyl methanesulfonate and ethyl methanesulfonate, while the rad2 null mutation enhanced the sensitivity of this system in response to ultraviolet (UV) radiation and a UV mimetic agent 4-nitroquinoline
1-oxide. In the rad2 null mutant, RNR3-lacZ induction was also more sensitive to MMS than that in wild type strain at low dosed. In summary, it appears that the enhancement of RNR3-lacZ induction is agent and repair specific, although inactivation of the nucleotide excision repair pathway may affect a broad range of testing agents which can be incorporated into the RNR3-lacZ test.
111 ACKNOWLEDGEMENTS
First, I would like to thank my supervisor, Dr. Wei Xiao for providing me with this interesting research project and invaluable guidance. His supervision of my work has proven to be a great help not only in the completion of this research but also in my other academic pursuits in the future. I have appreciated the opportunity of working with him over the years.
I would also like to thank the members of my supervisory committee, Dr. Barry
Blakley and Dr. Pat Krone for their support, helpful insights and useful suggestions for the progression of my research. A special thanks to Dr. Troy Harkness, my external examiner. Your presence set me at ease and made the experience enjoyable.
My gratitude is extended to all the members of Dr. Wei Xiao's laboratory: Yu
Zhu, Michelle Hanna, Parker Anderson, Carolyn Ashley, Leslie Barber, Landon
Pastushok, Stacey Broomfield, Todd Hysciw and Yu Fu, for their help and providing a friendly environment.
I greatly appreciate the financial support from NSERC grant obtained by Dr. W.
Xiao and scholarship support provided by the University of Saskatchewan.
A loving thanks to my husband and my parents, who gave me unconditional love, understanding, help and encouragement all the time. My gratitude cannot be expressed in words.
IV To my family
v Table of Contents
PERMISSION TO USE i ABSTRACT ii ACKNOLEDGEMENTS w DEDICATION v TABLE OF CONTENTS vi LIST OF TABLES ix LIST OF FIGURES x LIST OF ABBREVIATIONS xii
CHAPTER I: INTRODUCTION 1
1.1. DNA damaging agents and DNA damage 1 1.1.1. Endogenous DNA damages 2 1.1.2. Environmental DNA damaging agents 4 1.1.2.1. Physical agents that damage DNA 5 1.1.2.1.1. Ionizing irradiation 6 1.1.2.1.2. UV irradiation 6 1.1.2.2. Chemical agents that damage DNA 6 1.1.2.2.1. Alkylating agents 7 1.1.2.2.2. Base analogs 8 1.1.2.2.3. Chemicals that are metabolized to active forms 8
1.2. Cellular responses to DNA damage 10 1.2.1. DNA repair and DNA damage 11 1.2.1.1. Direct reversal DNA repair pathway 11 1.2.1.2. DNA excision repair 12 1.2 .1.2 .1. Base excision repair 12 1.2.1.2.2. Nucleotide excision repair 13 1.2.1.2.3. Mismatch repair 14 1.2.1.3. Recombination repair 15 1.2.1.4. Post-replication repair 16 1.2.2. Cell cycle checkpoints and DNA damage 17 1.2.3. Changes in gene expression after DNA damage 18 1.2.3.1. DNA damage inducible genes inS. cerevisiae 19 1.2.3 .1.1. DNA damage inducible genes that function directly in DNA repair 20 1.2.3 .1.2. DNA damage inducible genes that do not function directly in DNA repair 21 1.2.3 .1.2.1. RNR gene family and ribonucleotide reductase (RNR) 22 1.2.3 .1.2.2. Regulation of RNR3 gene expression 23
1.3.Current short-term genotoxicity testing systems 24
Vl 1.3.1. Tests that directly assess genetic alterations 25 1.3 .1.1. Bacterial reverse mutation test: the Ames test 25 1.3.1.2. In vitro mouse lymphoma thymidine kinase+/- gene mutation assay 28 1.3.1.3. Mammalian erythrocyte micronucleus test 29 1.3.2. Genotoxicity tests that assess cellular responses to DNA damage 30 1.3.2.1. SOS Chromotest 30 1.3 .2.2. Common reporters in genotoxicity assay 31 1.3 .2.2.1. Bacterial ~-galactosidase 31 1.3.2.2.2. Bacterialluciferase (LUC) 32 1.3.2.2.3. Green fluorescent protein (GFP) 33 1.3.2.2.4. Chloramphenicol acetyl transferase (CAT) 34
1.4. Development of a yeast RNR3-lacZ genotoxicity testing system 34 1.4.1. Advantages of yeast as host organism 36 1.4.2. Rationale of the present RNR3-lacZ testing system 37
1.5. Objectives of the present study 37
CHAPTER II: MATERIALS AND METHODS 39
2.1. Cell culture and manipulation 39 2.1.1. Yeast strains and cell culture 41 2.1.2. Yeast transformation 41 2.1.3. Yeast genomic and plasmid DNA isolation 39 2.1.4. ~-galactosidase (~-gal) activity assay 42 2.1.5. Chemicals and special media 43 2.1.6. Chemical and physical agents treatment 45 2.1.7. Cell killing 46
2.2. Molecular biology techniques 47 2.2.1. Bacterial culture and storage 47 2.2.2. Preparation of competent cells 47 2.2.3. Bacterial transformation 48 2.2.4. Plasmid DNA isolation (Mini-Prep) 48 2.2.5. Agarose gel electrophoresis 49 2.2.6. Isolation of DNA fragments from agarose gels 49 2.2.7. Ligation 50 2.2.8. Plasmids and plasmid construction 50 2.2.9. Radioactive labeling of DNA fragments 51 2.2.1 0. Southern Hybridization 54
CHAPTER III: CONSTRUCTION AND EVALUATION OF RNR3-IacZ SYSTEM 55
Vll 3.1. Introduction 55
3.2. Results 55 3.2.1. RNR3 is a more suitable reporter than other DNA damage-inducible genes 55 3.2.2. Induction of the RNR3-lacZ system by different agents 56 3 .2.3. Effects of incubation time on induction efficiency 59 3.2.4. Effects of cell growth stage on induction efficiency 59 3 .2.5. Creation and evaluation of a stable RNR3-lacZ testing system 64 3.2.5.1. Integration of RNR3-lacZ cassette into the yeast genome 64 3.2.5.2. Evaluation of the integrated RNR3-lacZ system 67
CHAPTER IV: ENHANCING THE SENSITIVITY OF RNR3-lacZ SYSTEM BY GENETIC MODICICATION OF THE HOST STRAIN 70
4.1. Introduction 70
4.2. Results 72 4.2.1. The effects of base excision repair mutations to RNR3-lacZ expression 72 4.2.2. Defection of nucleotide excision repair enhances the induction of RNR3-lacZ to UV radiation 79 4.2.3. Effects of recombination repair mutations on the induction of RNR3-lacZ 80 4.2.4. The RNR3-lacZ induction in PRR deficient mutant strains 87
CHAPTER V: DISCUSSION 91 5.1. Yeast system is suitable for genotoxicity testing 91
5.2. RNR3 gene is the best choice among yeast DNA damage-inducible genes 92
5.3. Quantitative assessment of the RNR3-lacZ testing system 93 5.3.1. Using of the "2-fold rule" to evaluate the results 93 5.3.2. Comparison of the RNR3-lacZ testing results with other genotoxicity testing systems 9 5 5.3 .3. The threshold of genotoxicity tests 96
5.4. Improvement of the RNR3-lacZ system 97 5.4.1. Alteration of host strains to enhance the sensitivity of RNR3-lacZ system 97 5.4.2. Considerations to develop a more user-friendly RNR3-lacZ system 99
REFERENCES 101
Vlll List of tables
Table Page
1-1. Comparison of commonly used p-gal, CAT, LUC and GFP reporters. 35
2-1. Yeast strains used in this study. 40
2-2. Agents used in this study and their mechanism of function. 44
2-3. Plasmids used in this study, their genotype and sources. 52
3-1. Summary of the RNR3-lacZ test and comparison with other test results. 62
IX List of figures
Figure Page
2-1. Integration of RNR3-lacZ cassette into HO locus. 53
3-1. Different response of RNRJ -lacZ, RNR2-lacZ and MAG 1-lacZ expression to MMS treatment. 57
3-2. The induction of RNRJ-lacZby different agents. 60
3-3. The response to RNR3-lacZ system to ethidium bromide. 61
3-4. Effects of incubation time on the induction efficiency of RNRJ-lacZ expression. 65
3-5. Effects of cell growth stage on induction efficiency of RNRJ-lacZ expression. 66
3-6. The genomic structure of integrated RNRJ-lacZ system was confirmed by Southern hybridization. 68
3-7. Comparison of RNRJ-lacZ induction between plasmid-borne and integrated RNR3-lacZ reporter. 69
4-1. Comparison ofMMS-induced RNR-lacZ expression in BER mutant strains and wild type DBY747. 75
4-2. Comparison of the RNRJ-lacZ induction in the magl null mutant and DBY747 after EMS treatment. 76
4-3. UV-induced RNRJ-lacZ expression in DBY747 and in mag] null mutant strain. 77
4-4. Comparison of the RNR3-lacZ induction in the magl null mutant, rad2 null mutant and the wild type DBY747 (c) after y-ray treatment. 78
4-5. Comparison of the RNR3-lacZ induction level in wild type DBY747 and that in the rad2 mutant after UV treatment. 81
4-6. Comparison of the RNR3-lacZ induction in wild type DBY747 and its rad2 null mutant after 4-NQO treatments. 82
4-7. Comparison of the RNRJ induction level in DBY747 and rad2 null mutant strain after MMS treatment. 83 4-8. Comparison of the RNRJ induction level in DBY747, rad50 and rad52 mutant strains after y-ray treatments. 85
4-9. Comparison of the RNR3-lacZ induction in the wild type DBY747 and rad52
X mutant strain after treatment with MMS. 86
4-10. Comparison of the RNR3-lacZ induction in DBY747 and rad6, rad18 mutant strains after MMS treatment. 89
4-11. Comparison of RNR3-lacZ induction in DBY747 and rad18 strain after UV radiation treatment. 90
XI List of Abbreviations
AAF N-2-Acety-2-Aminofluorene AP site apurinic or apyrimidinic site BER base excision repair f)-gal f)-galactosidase bp base pair CAT chloramphenicol acetyl transferase CHO Chinese hamster cell ddH20 double distilled water DMSO dimethyl sulfoxide EDTA ethylenediaminetetraacetic acid DSBs double stranded breaks E. coli Escherichia coli EMS ethyl methanesulfonate EtBr ethidium bromide FOA 5-fluoro-orotic acid GFP green fluorescent protein his histidine HU hydroxyurea HNPCC hereditary nonpolyposis colorectal cancer HPRT hypoxanthine-guanine phosphoribosyl transferase HR homologous recombination kb kilobase pair LUC luciferase MMS methyl methanesulfonate MMR mismatch repair MNNG N-methyl-N' -nitro-N-nitrosoguanidine mRNA messenger RNA NHEJ non-homologous end joining NER nucleotide excision repair 4-NQO 4-nitroquinoline OD optical density ONP 0-nitrophenol ONPG ortho-nitrophenyl-f)-galactoside PCR polymerase chain reaction PRR postreplication repair RNR ribonucleotide reductase SDmedium synthetic dextrose minimal medium S. cerevisiae Saccharomyces cerevisiae SDMH 1,2- dimethylhydrazine SSBs single strand breaks ssDNA single stranded DNA TK thymidine kinase Ubc ubiquitin-conjugating enzyme
Xll uv ultraviolet radiation XP Xeroderma Pigmentosum XPRT xanthine-guanine phosphoribosyl transferase
X Ill CHAPTER I: INTRODUCTION
1.1. DNA damage and environmental DNA damaging agents
A variety of toxicants are released into the general environment in large quantities and cause adverse health effects among people. Such effects are usually subclinical, except short-term responses that following acute exposure; however, cumulative damage also leads to chronic effects. Research indicates that a large proportion of human developmental diseases are caused by environmental agents that damage the genetic constitution of somatic cells. These agents, including chemicals, radiation, and viruses, are collectively referred to genotoxic agents. They can produce congenital malformation, cancer and a variety of more subtle effects on health and longevity.
Under normal conditions, four kinds of nitrogen bases (adenine, thymine, cytosine, and guanine) are contained in DNA molecules. Each base is held within the sugar phosphate backbone of the polynucleotide chain through an N-glycosylic bond. DNA helix formation relies on the proper hydrogen bonding between complementary bases on
opposite polynucleotide strands. Complementarity of bases matches cytosine with
guanine via three hydrogen bonds, and adenine with thymine via two hydrogen bonds.
It is assumed that this macromolecule must be extraordinarily stable in order to
maintain the high degree of fidelity. It is surprising to learn that the primary structure of
DNA is, in fact, quite dynamic and subject to constant change. Besides large-scale
changes such as chromosome rearrangement, translocation and transposition (Finnergan,
1990; Kleckner, 1990), DNA is also subject to alterations in the chemistry or the
sequence of individual nucleotides (Lindahl, 1993; Ward, 1985; Singer and Grunberger,
1 1982). Many of these changes arise as a consequence of errors introduced during replication, recombination, and repair itself. Other base alterations arise from the inherent instability of specific chemical bonds that constitute the normal chemistry of nucleotides under physiological conditions of temperature and pH. Surprisingly, DNA reacts readily with a variety of chemical compounds and physical agents both in vivo and in vitro.
DNA damage refers to the modifications of the DNA molecular structure or strand breaks. The damage will block replication and increase the probability of incorporation of incorrect bases during replication. Different agents, according to their chemical or physical characteristics, cause different types of DNA damage. The phenotypic result of
DNA damage may depend on the chromosomal locus at which it occurred, and on how the base sequence is affected. Some of the DNA damaging agents can be produced via cell metabolism, but most of them are present in the environment. For convenience,
DNA damage can be classified into two major groups, referred to as endogenous DNA
damage and exogenous DNA damage.
1.1.1. Endogenous DNA damage
DNA lesions can arise naturally through intracellular metabolism or the intrinsic
instability of DNA. The chief source of DNA alterations arising during normal DNA
metabolism is the mispairing of bases during DNA synthesis that results in base
mismatches. Mismatches can arise from the synthetic events associated with a subset of
nuclear DNA during repair and recombination. Generally, all of the bases in a genome
must be replicated prior to cell division. However, under certain circumstances the
2 replication of DNA occurs on parental template strands carrying unrepaired noninstructional or misinstructional base damage, which can act as miscoding lesions.
Each of the common bases in DNA can spontaneously undergo a transient rearrangement of bonding to form a structural isomer of the base. Formation of an isomer of any base alters its base-pairing properties. If the base isomer persists during replication, DNA polymerase may incorporate incorrect bases opposite the modified base. If the incorporated base of the daughter strand remains, the lesion is fixed into a mutation.
Deamination of cytosine, adenine and guanine is also a major source of mutation.
Deamination of cytosine, resulting in the formation of uracil, is the most common deamination reaction in the cell (Lindahl, 1993). The conversion of cytosine to uracil often results in a transition mutation, since adenine is likely incorporated opposite uracil.
The base 5-methylcytosine, is a naturally occurring analogue of cytosine. The deamination of 5-methylcytosine causes the conversion from cytosine to thymine and therefore produces a transition mutation. The deamination of adenine, and guanine results in products of hypoxanthine and xanthine respectively. Hypoxanthine is a potentially mutagenic agent. Xanthine can arrest DNA replication and therefore cause cell death
(Friedberg et al., 1995).
The loss of purines and pyrimidines from DNA has been most extensively studied at acidic pH. However, depurination and depyrimidination can also occur at appreciable rates at neutral or alkaline pH (Lindahl, 1993; Loeb and Preston, 1986). The chemical mechanism of hydrolytic DNA depurination at acidic pH is believed to be the same as that established for acid hydrolysis of deoxynuclosides. The mechanism of hydrolysis of
3 nucleosides at neutral and at alkaline pH is less well characterized. In E. coli, the rate of depurination is about one purine per cell per generation at 37 °C. For mammalian cells, in which genomes are much larger and replication times are longer, the loss of purine is estimated at the rate of 10,000 per cell per generation. Pyrimidine nucleosides are more stable than purine nucleosides with respect to the glycosylic linkage of the bases to deoxyribose. The mechanism of depyrimidination is the same as for depurination, but cytosine and thymine are lost at rates only 1/20 of that for adenine or guanine. This still results in the loss of hundreds of pyrimidines per mammalian cell per generation.
Attack by reactive oxygen species is another major source of spontaneous damage to DNA and other intracellular macromolecules such as proteins, lipids, and carbohydrates (Joenje, 1989; Ames and Gold, 1991 ). Reactive oxygen species are created during aerobic metabolism. The major intracellular source of oxygen radicals is probably leakage associated with the reduction of oxygen to water during mitochondrial respiration. These products include single oxygen, peroxide radicals (02-), hydrogen peroxide (H202), and hydroxyl radicals <-OH). They create single and double strand breaks, damage to the deoxyribose moiety, lethal abasic sites and various base modifications (Croteau & Bohr, 1997). The lesions induced by these reactive oxygen species overlap with those induced by ionizing radiation, since much of ionizing radiation-induced DNA damage is caused by reactive oxygen species.
1. 1. 2. Environmental DNA damaging agents
The environment outside a cell or organism contains many natural and artificial
4 DNA damaging agents. According to their sources, they can be classified as physical or chemical agents.
1. 1. 2. 1. Physical agents that damage DNA
Physical DNA damaging agents are generally referred to as ionizing irradiation and
UV irradiation.
1. 1. 2. 1. 1. Ionizing irradiations
Ionizing radiation has been a source of naturally occurring physical damage to the
DNA of living organisms since the beginning of biological evolution. However, various man-made therapeutic, diagnostic, or occupational sources of exposure to ionizing radiation are of far greater importance. In 1926, Muller exposed fruit flies to X rays and showed for the first time that ionizing radiation produces mutations within individual genes as well as gross chromosomal aberrations. Ionizing radiation can randomly cause damage to all cellular components and induces a variety of DNA lesions. Radiation damage to DNA is ascribed to both direct and indirect effects. Direct effects result from the direct interaction of the radiation energy with DNA. Indirect effects result from the interaction of DNA with reactive species formed by the radiation. The lesions created by ionizing radiation include base damage, sugar damage and strand breaks. It has been estimated that damage to sugar residues in DNA may be less than damage to bases.
However, such damage is of biological importance primarily because of the strand breakage that might result. Ionizing radiation also induces strand breakage directly, and most of the lethal effects of ionizing radiation can be attributed to these lesions
5 1. 1. 2. 1. 2. Ultraviolet irradiation
Ultraviolet light is highly relevant biologically. Living organisms have had to contend with the genotoxic effects of solar UV radiation since the beginning of evolution of life on this planet. It has been suggested that unattenuated UV radiation during the pre-Phanarazoic aeons might have precluded the development of terrestrial life. At present, the exposure of cells to UV radiation is probably the best studied and most extensively used model system for exploring the biological consequences of DNA damage, its repair and tolerance. The UV radiation spectrum has been subdivided into three wavelength bands designated UV -A ( 400 to 320 nm), UV -B (320-290 nm), and
UV-C (290-IOOnm). Solar UV radiation consists mainly ofUV-A and UV-B, so they have more relevance to human health. Most of UV -C is absorbed by the atmospheric ozone layer. When DNA is exposed to radiation at wavelengths approaching its absorption maximum (---260 nm), adjacent pyrimidines become covalently linked by the formation of a four-membered ring structure, called cyclobutane dipyrimidine or pyrimidine dimer. Some photoproducts such as pyrimidine-pyrimidine (6-4) lesions are also present in UV -irradiated DNA. These photoproducts introduce a major distortion in the double helical structure of DNA. Other photoproducts in DNA include pyrimidine hydrates, thymine glycols, but they are not very common (Friedberg et al. 1995).
1. 1. 2. 2. Chemical agents that damage DNA
Chemical substances that induce mutation occur in widely divergent chemical groups, ranging from simple compounds such as formaldehyde to complex ones such as alkaloids. Many of these chemicals are carcinogenic. Exposure to chemicals is
6 ubiquitous in industrial societies. It has been estimated, for example, that more than
50,000 chemicals are in common use in the United States. Most of these compounds have not been tested for mutagenicity; of those that have been, about 20 percent produced mutations in the Ames test. The total exposure to mutagenic chemicals is thus unknown, but it undoubtedly presents a definite public health risk.
1. 1. 2. 2. 1. Alkylating agents
Alkylating agents are electrophilic compounds with affinity for nucleophilic centres in organic macromolecules. These include a wide variety of chemicals, many of which are proven or suspected carcinogens. There are two types of alkylating agents: monofunctional and bifunctional. The former has a single reactive group and thus interacts covalently with single nucleophilic centres in DNA. Bifunctional alkylating agents have two reactive groups. Each molecule is potentially able to react with two sites in DNA. There are numerous potential reaction sites that can be alkylated within a DNA
1 7 1 strand. For monoalkylating agents, these sites include: N , Jt, !I, N in adenine; N , N-,
3 7 6 2 4 3 N , N , and 0 guanine; N , d, and 0 in thymine; and N , !I, and d in cytosine.
Alkylation of oxygen in phosphodiester linkages results in the formation of phosphotriesters (Singer, 1986). Base modification by alkylation weakens theN glycosylic bond. Hence treatment with many alkylating agents leads to depurination/depyrimidination and the appearance of alkali-labile abasic sites (Friedberg et al., 1995).
Bifunctional alkylating agents can react with two different nucleophilic centres in
DNA. If the two sites are on opposite polynucleotide strands, interstrand DNA cross-
7 links occur. If these sites are situated on the same polynucleotide chain of a DNA duplex, the reaction product is referred to as an intrastand cross-link. Interstrand DNA links represent an important class of chemical damage to DNA, since they prevent DNA strand separation and hence can constitute complete blocks to DNA replication and transcription. Nitrogen mustard, mitomycin and various platinum derivatives are famous cross-linking agents (Lawley and Phillips, 1996). In addition, UV radiation at 254 nm and ionizing radiation can result in the formation of intermolecular DNA cross-links as well as DNA-protein cross links as minor products of DNA damage (Friedberg et al.,
1995).
1. 1. 2. 2. 2. Base analogs
Generally, the biologically relevant base damage does not originate from the direct reaction of chemical agents with DNA itself, but from the incorporation of damaged deoxynucleotides during replication of damaged templates. Among the most extensively studied base analogs are the halogenated uracil derivatives, 5-bromouracil, 5-fluorouracil, and 5-iodouracil. All of them are thymine analogs that can result in mutations when present in template DNA undergoing DNA replication (Miller, 1983; 1985; 1992). The adenine analog, 2-aminopurine is also mutagenic (Rotilio, 1986; Sowers et al., 1987).
1. 1. 2. 2. 3. Chemicals that need to be metabolized to active forms
A variety of relatively nonpolar (and hence chemically unreactive) compounds can be metabolized to more reactive forms in the affected cells. Many of these compounds are potent mutagens and carcinogens. It is now known that the metabolic activation of
8 these compounds is catalyzed by specific metabolizing enzymes. The biological function of these enzyme systems is to protect cells against cytotoxic effects by converting potentially toxic nonpolar chemicals to water-soluble excretable forms. However, some of the reaction products become activated to electrophilic forms that are particularly reactive with nucleophilic centres in organic macromolecules such as DNA. The best studied activating enzymes are the cytochrome P-450 system (Ullrich, 1977). Other examples involved in activating genotoxic chemicals include microsomal and cytoplasmic glutathione-S-transferases, sulfotransferase, acetyltransferases, UDP glucuronosyltransferases, and adenosylating as well as methylating enzymes.
N-2-Acetyl-2-Aminofluorene (AAF) belongs to a class of chemicals known as aromatic amines. After being catalyzed by cytochrome P-450 and cytosolic enzymes, it becomes a highly reactive alkylating agent (Friedberg, 1995).
Aflatoxin, especially Aflatoxin B 1, is one of the most potent liver carcinogens. It is oxidized by the mixed function oxygenases of the P-450 system in liver cells, and gives rise to aflatoxin B 1-8, 9-epoxide as the major product. Certain guanine residues in double-stranded DNA are preferentially attacked by this reactive electrophilic epoxide
(Friedberg, 1995).
4-Nitroquinoline 1-0xide (4-NQO) is often referred to as a "UV radiation mimic" agent because it produces bulky adducts. The repair of these bulky adducts by nucleotide excision repair (NER) is similar to the repair of cyclobutane pyrimidine dimer and (6-4) photoproducts. Seryl-tRNA-synthetase is known involved in the activation of 4-NQO.
Benzo(a)pyrene is a very potent carcinogenic polycyclic aromatic hydrocarbon that has a remarkable correlation with cancer of the scrotum. Unmodified benzo(a)pyrene is
9 an unreactive nonpolar compound. It is now known that components of the P-450 system known as arylhydrocarbon hydroxylases can metabolize benzo(a)pyrene to excretable phenols and dihydrodiols. Some of the metabolites are electrophilic epoxides that can damage DNA.
Glutathione is a major intracellular sulfhydryl component. It is strongly nucleophilic and reacts with electrophilies. Glutathione conjugation is, in general, considered to be a detoxification reaction. However, it can be potentially genotoxic when it involves the activation of carcinogenic agents such as N-nitrosamines (Singer and
Grunberger, 1983) and N-methyl-N' -nitro-N-nitrosoguanidine (MNNG) (Kistler et al.,
1986).
1.2. Cellular response to DNA damage
When cells are exposed to conditions that cause DNA damage, they activate numerous repair mechanisms that result in the restoration of the DNA sequence to its original condition. The repair mechanism involves various overlapping repair pathways.
However, if the cell is exposed to conditions that result in severe DNA damage and/or conditions that interfere with chromosomal replication, it co-ordinately activates the expression of a large number of diverse unlinked genes involved in DNA repair and error-prone DNA replication. This coordinated activation of diverse metabolic functions
in response to DNA damage has been called the SOS response in prokaryotes. Similar
mechanisms have also been found in eukaryotic cells. Cellular responses that do not
include the removal of the primary damage to the genome are considered DNA damage
tolerant rather than DNA repair mechanisms. Many DNA tolerance mechanisms result in
10 permanent mutations in the genome. The ability of cells to tolerate DNA damage is biologically as important as their ability to repair such damage.
1.2.1. DNA repair
DNA repair is involved in processes that minimize cell killing, mutations, replication errors, persistence of DNA damage and genomic instability. Abnormalities in these processes have been implicated in cancer and aging. The robust DNA repair systems are necessary for enhanced survival and reduced mutagenesis of cells. DNA repair includes direct reversal of DNA damage, DNA excision repair, DNA recombinational repair and post -replication repair pathways.
1.2.1.1. Direct reversal of DNA damage
The direct reversal DNA repair pathway removes the lesion without additional alteration to the native DNA molecules. This type of DNA repair is often performed by a single enzyme that only recognizes the specific lesions. The repair is usually just a single-step reaction that restores the DNA structure to its normal state.
As indicated in section 1.1.2.1, cyclobutane pyrimidine dimer and pyrimidine (6-4) pyrimidine photoproducts are the two major classes of cytotoxic, mutagenic and carcinogenic DNA photoproducts produced by UV light irradiation of cells. The dimer is lethal to the cell if it is not removed before replication (Berends et al., 1961 ). Direct reversal of DNA repair pathway which removes pyrimidine dimers (Nairn et al., 1989;
Sancar, 1990) and (6-4) photoproduct (Todo et al., 1993) have been reported, respectively.
11 Another example of direct reversal of DNA damage is found in the repair of alkylation damage. Certain alkylating lesions can be removed by the alkyltransferase family. Alkyltransferases have been isolated in bacteria, yeast and mammalian cells.
They can directly remove the alkyl group from DNA molecules by accepting the alkyl group at a cysteine residue in their active sites (Pegg and Byers, 1992). For example, the
0 6 -alkyl guanine DNA alkyl transferase (Mgt1) inS. cerevisiae can specifically remove the methyl group from 0 6-methyl guanine and 0 4 -methythymine lesions (Xiao et al.,
1991).
1.2.1.2. DNA excision repair
DNA excision repair consists of at least three separate pathways: base excision repair (BER), NER and the mismatch repair pathway (MMR, Friedberg et al., 1995).
These pathways remove DNA lesions by excising the damaged sequence, followed by replacement with undamaged nucleotide sequence.
1.2.1.2.1. Base excision repair
All BER is initiated by the action of a specific class of DNA enzymes called DNA glycosylases. The glycosylase recognizes and binds to the damaged site, then mediates the cleavage of the damaged base from the sugar backbone. The resulting abasic site is incised exclusively by AP endonucleases. The one-nucleotide gap is filled by DNA polymerase and sealed by DNA ligase (Wallace, 1994; Sancar, 1994; Seeberg, 1995).
This process is called short-patch BER. An alternative BER pathway, which is called long-patch BER, has been discovered in eukaryotes that involves the replacement of
12 more than a single nucleotide (about 7 nucleotides). In long patch BER, DNA polymerase fills in the missing base and continues to displace the existing strand. The resulting flap-like structure is excised by Fen1/ Rad27, and the strand break is ligated together (Wilson and Thompson, 1997).
Defects in BER have been shown to result in hypersensitivity to alkylating agents, oxidative agents, ionizing radiation and endogenous damage (Wallace, 1994). This suggests that BER is an important pathway for protecting cells from mutagenic and lethal damage.
1.2.1.2.2. Nucleotide excision repair
The NER pathway primarily repairs DNA damage resulting from UV, 4-NQO and cross-linking agents. All these DNA damaging agents cause helix-distorting lethal lesions. The following steps are involved in NER: lesion recognition, strand separation between damaged and undamaged strands, excision of the damaged sequence and polymerization of DNA to replace the excised sequence (Friedberg et al., 1995).
There are two classes of NER: transcription-coupled NER and global genome NER.
Studies carried out with E. coli, yeast and mammalian cells demonstrate that NER preferentially repairs actively transcribed genes (Bohr and Kober, 1985; Mellon and
Hanawalt, 1989~ Smerdon and Thoma, 1990). The preferential repair of active genes is accounted by the fast repair of the transcribed strand. In contrast, NER also can recognize and repair lesions throughout the genome, but this random process occurs slowly.
13 Lacking of XP genes and therefore, a defective in NER pathway results in a human hereditary disease known as Xeroderma Pigmentosum (XP; Cleaver, 1968). One of the most consistent cellular phenotypes ofXP cells is an increased sensitivity to killing following exposure to a wide variety of DNA damaging agents, including UV radiation.
Patients with XP syndrome have an extremely high incidence of premalignant actinic keratoses, as well as benign and malignant skin tumors (Friedberg et al., 1995).
1. 2. 1. 2. 3. Mismatch repair pathway
Three different components contribute to the overall fidelity of DNA replication: first is the polymerization reaction itself; second is the proofreading by a 3 '~ 5' exonuclease acitivity; finally, the postreplication mismatch repair. Without the intervention of any cellular factors, the error frequency during DNA replication is about
1o- 1~ 1o- 2 in E. coli (Loeb and Kunkel, 1981 ). The combined effects of base selection and proofreading of newly inserted nucleotides by certain DNA polymerases, together with the enhancement of DNA synthesis reduce the error efficiency by 3-6 orders of magnitude. Mismatch repair increases the error frequency to approximately 10-10
(Kunkel and Loeb, 1981 ).
Heritable mutations in a MMR gene lead to a so-called mutator phenotype, that is a very high susceptibility of the cell or organism to mutations (Loeb, 1994). It is known that hereditary nonpolyposis colorectal cancer (HNPCC) is resulted by defects in MMR gene hMSH2 or hMLH1. A striking feature of HNPCC is instability of simple repeated sequences (Fischer et al., 1992; Parsons et al., 1995).
14 1. 2. 1. 3. Recombination repair
The induction of double strand breaks (DSBs) in DNA by exposure to DNA damaging agents, or as intermediates in normal cellular processes, constitutes a severe threat for the integrity of the genome. If not properly repaired, DSBs may result in chromosomal aberrations, which, in tum, can lead to cell death or to uncontrolled cell growth. To maintain the integrity of the genome, multiple pathways for the repair of
DSBs have evolved during evolution: homologous recombination (HR), non-homologous end joining (NHEJ) and single-strand annealing (SSA). Homologous recombination has the potential to lead to accurate repair ofDSBs, whereas NHEJ and SSA are essentially mutagenic. Homologous recombination is the common way for repair DSB inS. cerevisae, but in higher eukaryotes, both HR and NHEJ are important. InS. cerevisiae, the RAD5 2 epistasis group genes are responsible for recombination repair. This group includes RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, RDH54/TIDJ,
MREJJ, and XRS2 (Friedberg et al., 1995). S. cerevisiae is extremely efficient at catalyzing homologous recombination. It involves interactions between two DNA molecules sharing substantial homology, and includes both reciprocal and nonreciprocal information exchange. Reciprocal exchange does not alter the content of the genome but rearranges genetic linkage patterns. Nonreciprocal exchange results in a net gain or loss of functional information. When a DSB occurs, it is resected by 5' to 3' exonucleases to expose single-stranded tails with 3' termini. This single stranded tail invades an uncut homologous duplex and promotes repair synthesis followed by branch migration to produce two Holliday junctions. The Holliday junctions are then resolved to the normal structure of DNA strand by Holliday junction resolvases (Friedberg et al., 1995). This
15 progress ensures that all missing sequence is replaced by replicating from the homologous duplex molecule sequence (Shinohara and Ogawa, 1995). Rad51
(homologous to E. coli RecA) assembles on the 3 '- ended single-stranded DNA and, in concert with Rad54, stimulates strand invasion at homolgous sequence and facilitates the initiation of DNA synthesis (Paques and Haber, 1999). Other proteins, such as Rad52,
Rad55 and Rad57, are believed to assist the functions ofRad51 and Rad54 (Sung, 1997).
Although RAD52 group genes share the property of conferring resistance to ionizing radiation, they show considerable variation in assays for DSB repair and recombination. RAD5 0, XRS2, and MREJJ form a subgroup with similar properties.
Their complex is nucleolytic and plays a role in nonhomologous end rejoining/filling
(Moore and Haber, 1996).
1. 2. 1. 4. Post-replication repair
DNA post-replication repair (PRR) is defined as an activity to convert DNA damage-induced single-stranded gaps into large molecular weight DNA without actually removing the replication-blocking lesions. Hence, PRR is actually a damage tolerance pathway. In bacteria such as E. coli, this activity requires RecA and the RecA-mediated
SOS response and is accomplished by recombination and mutagenic translesion DNA synthesis. In the yeastS. cerevisiae, PRR is centrally controlled by RAD6 and RAD18, whose products form a stable complex with single-stranded DNA-binding, A TPase and ubiquitin-conjugating activities. Post-replication repair can be further divided into translesion DNA synthesis and error-free modes. Mutagenic translesion bypass is due to the mutagenic DNA polymerases, such as Pol~ and Poll") in yeast; hRev3, XPV, Poh and
16 PolK in human; UmuCD and DinB in E. coli (Baynton and Fuchs, 2000; Goodman and
Tippin, 2000; Johnson et al, 2000). In the yeastS. cerevisiae, error-free PRR requires
Rad6, Rad18, Mms2, Ubc13, Rad5, Pol30 (PCNA) and Pol13 (Pol8). The PRR processes appear to be highly conserved within eukaryotes, from yeast to human
(Broomfield et al., 2001 ).
1. 2. 2. Cell cycle checkpoints and DNA damage
DNA lesions can be fixed in the genome of a cell in the form of a mutation, that is, a heritable alteration that can be corrected only by elimination of the cell. Genetic stability is controlled by a number of interrelated and overlapping cellular functions, the most important of which are the various DNA replication, repair and recombination pathways. A major component of cellular defense against DNA damage are cell cycle checkpoints, which are monitoring systems for DNA damage that temporarily halt replication until the lesions are repaired (Carr, 1996). These mechanisms are thought to prevent the replication of damaged templates and the segregation of broken chromosomes. The DNA damage checkpoint pathway is well conserved from yeast to humans. The following genes: RAD9, RAD17, RAD24, RAD53, CHKJ, DCDJ, DCD2,
MEC3, POL2, and MECJ have been identified in the yeastS. cerevisiae. When mutated, these genes inactivate certain checkpoint controls (Elledge, 1996; Paulovich et al., 1997), among which~ RAD9, 17, 24 and MEC3 are required to activate the DNA damage checkpoint when cells are in G 1 or G2 phase (Hartell and Weinert, 1989; Siede et al.,
1993; Weinert and Hartwell, 1994); POL2 is required to sense replication blocks when cells are in S phase (Navas et al., 1995); whereas MECJ (Paulovich and Hartwell, 1995)
17 and RAD53 (Allen et al., 1994) appear to form a downstream signal transduction cascade required for all three cell cycle stages.
In mammalian cells, two important examples of cell cycle checkpoint components are the tumor suppressor genes p53 and ATM The p53 gene is activated by DNA damage to subsequently induce the p21 cdk inhibitor, which mediates growth arrest
(Hartwell and Kastan, 1994). The p53 gene product is also considered to play a key role in apoptosis pathways (Lowe et al., 1993). The ATM protein might be an upstream component of a radiation-induced signal transduction pathway which is activated by
DNA damage and involves the recruitment of p53 (Savitsky et al., 1995), and perhaps other checkpoint proteins (Carr, 1996). The ATM protein shares the phoshoinositide 3- kinase (PI-3 kinase) domain with several large proteins in yeast, Drosophila, and mammals (Keith and Schreiber, 1995). There is also significant homology between ATM and the yeast cell cycle checkpoint gene, MECJ.
1. 2. 3. Changes in gene expression after DNA damage
In response to DNA damage, both prokaryotic and eukaryotic cells activate stress responses that result in specific alterations in patterns of gene expression and active inhibition of cell division.
The SOS regulatory network of E. coli is a stress response that is well understood.
Exposure to a variety of DNA-damaging agents induces the expression of approximately
30-40 genes through a common mode of transcriptional de-repression controlled by the recA and lexA gene products. As a consequence of DNA damage or interference with
DNA synthesis, single-stranded DNA accumulates and activates RecA molecules.
18 Activated RecA potentiates the autoproteolytic cleavage of the LexA repressor that binds within the operators of SOS-inducible genes that mediate a number of functions related to
DNA metabolism and cell division. Similar stress responses have been found inS. cerevisiae and mammalian cells although it is much more complex than that in E. coli.
The transcription of a number of yeast genes has been demonstrated to accumulate after exposure to DNA-damaging agents, and at least some of them function directly in DNA repair. Some regulatory genes that control the transcriptional response to DNA damage have been identified. Interestingly, some of these regulatory genes are also functional in cell-cycle checkpoint controls that couple cell-cycle progression to the status of genome duplication and integrity. Interfering with DNA replication activates the S-phase checkpoint that prevents entry into mitosis with unreplicated DNA. DNA lesions encountered outside of S phase activate additional checkpoint pathways that delay cell cycle progression prior to the G 1/S and G2/M transitions, presumably to prevent the replication or segregation of damaged chromosomes.
1. 2. 3. 1. DNA damage inducible genes in S. cerevisiae
The number of genes induced by DNA damage in S. cerevisiae is very large. In a recent genome scale study (Jelinsky and Samson, 1999), as many as 325 genes showed at least a 4-fold increase at the transcription level after treatment with the alkylating agent
MMS. This represents about 5o/o of the yeast genome. These genes can be divided into several classes, some of them function directly in the repair of damaged DNA, and some others function primarily in nucleotide metabolism, DNA synthesis and protein turnover.
19 1. 2. 3. 1. 1. DNA damage inducible genes that function directly in DNA repair
Within the NER pathway, RAD2, RAD27, and RAD23 are induced approximately five fold by UV irradiation (Madaua and Prakash, 1986; Robinson et al., 1986; Jones et al., 1990). RAD2 encodes an endonuclease and is required for the incision step ofNER.
RAD7 and RAD23 appear to fulfill accessory or regulatory roles inNER biochemistry. In addition to UV irradiation, RAD2 also responds to other DNA damaging agents such as
4-NQO, y irradiation, bleomycin, nitrogen mustard, mitomycin, MMS and cyclohexamide (Robinson et al. 1986).
Within the recombinational repair pathway, RAD51 and RAD54 are DNA damage inducible. Both of them also function in normal recombination. Their expression can be induced 5-10 fold by X ray irradiation and MMS (Cole et al., 1987; Basile et al., 1992).
Within the PRR pathway, RAD6 encodes a ubiquitin-conjugating enzyme (Ubc) that is required in response to UV irradiation. RAD18 encodes a protein with DNA binding domains that is capable of binding single-stranded DNA. Research (Bailly et al., 1994) indicated that Rad6 and Rad 18 form a heterodimer whose function is similar to that of
RecA in E. coli. Rad18 binds single-stranded DNA formed at a stalled replication fork and targets Rad6 to the site of damage. Both RAD6 and RAD18 can be induced to several folds by UV irradiation (Madura et al., 1990; Jones and Prakash, 1991 ). Rad30 is a newly discovered DNA polymerase (Pol11) and is placed in the error-free arm of PRR
(Johnson et al., 1999). It is also UV inducible (McDonald et al., 1997)
MAGI encodes a 3'-methyladenosine DNA glycosylase specifically involved in the repair of alkylated bases (Chen et al., 1989). It is induced not only by alkylating agents
20 like MMS and MNNG, but also by UV irradiation and 4-NQO (Chen et al., 1990; Chen and Samson, 1991).
PHRJ encodes the yeast photoreactivating enzyme that catalyzes the light dependent repair of pyrimidine dimer in DNA and stimulates NER activaty in the dark.
PHRJ is specific for the repair of pyrimidine dimer, but is induced by a variety of DNA damaging agents by chemically modifying DNA or causing DSBs (Sebastian et al.,
1990).
The mechanism of why and how these genes are induced is still not completely understood. Some genes can be induced by a variety of DNA damaging agents. For example, generally, UV damage does not need 3-methyladenine DNA glycosylase for the repair, but MAGI is also induced by UV irradiation. It has been postulated that the promiscuous inducibility of genes occurs either because the sensory/signalling system for inducible repair genes can recognize several forms of DNA damage, or because different forms of DNA damage are converted into discrete form or intermediate that acts as a damage signal (Nickoloff and Hoekstra, 1998).
1. 2. 3. 1. 2. DNA damage inducible genes without direct function in DNA repair
There are a number of other inducible genes that do not function directly in the above repair pathways. Most of them function in the metabolism of nucleic acids or proteins. For example, CDC8 encodes thymidylate kinase, CDC9 encodes DNA ligase,
HIS3 and HIS4 function in histidine metabolism. The best studied gene family is the
RNR gene family.
21 1. 2. 3. 1. 2. 1. RNR gene family and ribonucleotide reductase (RNR)
The RNR family consists of RNRJ, RNR2, RNR3 and RNR4. They encode ribonucleotide reductase (RNR) which catalyzes the first and rate-limiting step in the production of deoxyribonucleotides. The reaction is of the following form:
NTP + reductant-(SH)~ dNTP + reductant-(S-S)
Deoxyribonucleotide levels are critical to many cellular functions. During the DNA synthetic phase of the cell cycle, their levels must be sufficient to replicate the genome.
Furthermore, their relative ratios must be balanced to ensure high-fidelity DNA replication. Unbalanced nucleotide pools lead to enhanced misincorporation and mutation frequency. Deoxyribonucleotide levels are also important for DNA repair processes. All of excision repair, recombination repair and PRR require DNA synthesis and are thought to be impaired in the absence of sufficient nucleotide levels.
Ribonucleotide reductase is an enzyme with a 2P2 structure. RNRJ and RNR3 encode the alternative large subunit Rl. RNRJ encodes al and RNR3 encodes a'.
Normally, Rl is a dimer of al with a monomeric molecular mass of90 kDa (Caras et al.,
1985). Each monomer contains two distinct binding sites for deoxynucleoside triphosphates which act as allosteric regulators of the enzymatic activity. One site controls the substrate specificity of RNR enzyme and is responsible for maintaining balanced nucleotide pools, thus facilitating optimal fidelity of DNA replication. The other monitors the ATP/dA TP ratio and modulates the overall activity of the enzyme, presumably to ensure that sufficient dNTPs are produced for DNA synthesis without depleting ribonucleotides needed for RNA synthesis. RNR2 and RNR 4 encode the small subunit R2 which contains a binuclear ferric iron centre and a tyrosyl free radical that is
22 essential for activity. RNRJ, RNR2, and RNR4 are essential for mitotic growth while
RNR3 is not. It is assumed that under normal vegetative conditions, only RNRJ, RNR2 and RNR4 are expressed, and thus the ribonucleotide reductase in the cell is a2PP'.
However, in the presence of DNA damage, RNR3 is induced, producing other forms of the enzyme (Huang and Elledge, 1997).
The expression of RNRJ and RNR2 is increased in response to UV light, the UV mimetic compound, 4-NQO, bleomycin, and MMS. They can also be induced by an inhibitor of ribonucleotide reductase - hydroxyurea (HU) which functions by quenching the free radical on the small subunit, thus inhibiting the activity of RNR enzyme
(reviewed by Elledge et al., 1992). The induction of RNR3 can reach 50-100 fold by
MMS and 4-NQO and 50-200 fold by UV irradiation (Yagle and McEntee, 1990; Elledge and Davis, 1990). Why can RNR3 be induced to such so high levels? Since its function and regulatory mechanisms are highly conserved, it is hypothesized that RNR3 must provide a selective advantage, but so far the nature of this advantage remains unclear.
1. 2. 3. 1. 2. 2. Regulation of RNR3 gene expression
Both expression and activity of ribonucleotide reductase are highly regulated. The
RNR genes are cell cycle-regulated and inducible by DNA damaging agents in all organisms examined, including yeast (Elledge and Davis, 1987; Hurd, et al., 1987;
Elledge and Davis, 1990) and human (Hurta and Wright, 1992). In addition to high levels of expression in S phase, RNR genes are further up-regulated when DNA synthesis is blocked.
23 The expression of RNR3 is under the negative regulation by Crt 1 which is homologous to the mammalian RFX family of DNA binding proteins that can bind to a conserved DNA sequence, the "X box". The X box sequences have been identified in the promoter of RNR3 and other RNRs (Huang et al., 1998). It is a highly conserved DNA sequence of 13 nucleotides firstly found in the promoter of all MHC class II genes (Reith et al., 1990). One strong X box and two weak boxes are found in the RNR3 promoter.
Under normal conditions, Crt1 binds to the X box and recruits the Ssn6-Tup1 repression complex to the promoter region, thus repressing the transcription of RNR3. Two models have been proposed to explain the repression by the Tup 1-Ssn6 complex. The first model suggests that the complex may inhibit transcription through direct interaction with one or more of these transcription factors. In the other hypothesis, the Ssn6-Tup 1 complex exerts repression through modulation of chromatin structure. The activation of the
Mec1-Rad53-Dun1 pathway involves inactivation ofCrt1 by phosphorylation. As Crt1 becomes phosphorylated in response to the replication blocks and DNA damage, it loses its ability to bind to X-box elements and Ssn6-Tup1 complex is released. This leads to transcriptional induction of RNR3 (Huang et al., 1998).
1. 3. Current short-term genotoxicity testing systems
Many chemical and physical methodologies have been developed to meet the greater demand to detect environmental pollutants. Some of them require analytical equipment such as gas or high-pressure liquid chromatography, mass and atomic absorption spectrometry. Although they are powerful, accurate and sensitive, they are expensive and need to be run in specialized laboratories. More importantly, they fail to
24 provide data as to the bioavailability of a pollutant, the effects of pollutant on living systems, and the potential interaction (synergistic/antagonistic) among different toxicants.
To overcome these disadvantages of chemical and physical methods, a variety of bioassays have been developed.
Genotoxicity testing is a key element for safety evaluation of food, drugs, as well as environmental health. Genotoxicity tests are in vitro and in vivo tests designed to detect compounds that induce genetic damage. A large number of genotoxicity tests are presently available for use in hazard evaluation, including: ( 1) tests that directly assess the genetic alterations (gene mutations and chromosomal effects), and (2) indirect genotoxicity tests that respond to DNA damage known to lead to these alterations. The latter category of tests may assess either DNA damage (e. g., DNA adducts or DNA strand breakage) or cellular responses to DNA damage (e. g., unscheduled DNA synthesis).
1. 3. 1. Tests that directly assess genetic alterations
Some experiments are designed to detect the alterations on DNA and chromosomes.
The most commonly used tests are listed below.
1. 3. 1. 1. Bacterial reverse mutation test: the Ames test
It is difficult, time consuming and expensive to directly assay potential carcinogens by testing their ability to form tumors in animals. However, in addition to causing tumors in animal cells, most carcinogens are mutagens. Based upon this insight, Ames and colleagues (Ames et al., 1973a; b) developed a simple, indirect assay for potential
25 carcinogens. The assay is based upon the reversion of mutations in the histidine (his) operon in the bacterium Salmonella typhimurium. The his operon encodes enzymes required for the biosynthesis of the amino acid histidine. Strains with mutations in the his operon are histidine auxotrophs and are unable to grow in the absence of histidine.
Revertants that restore the His+ phenotype will grow on minimal medium plates without histidine. This provides a simple, sensitive selection for revertants of his mutants.
The his mutants are mixed with the potential mutagen and then plated on minimal medium with a very small amount of histidine. The concentration of histidine used is limiting, so that after the cells go through several cell divisions, the histidine is used up and the auxotrophs stop growing. However, if the potential mutagen induces His+ revertants during the initial few cell divisions, each of the resulting revertants will continue to divide and form a colony. The number of colonies produced is proportional to how efficiently the mutagen reverts the original mutation. For example, if chemical A produces a higher frequency of reversion than the control, it is considered a mutagen and likely a carcinogen. If chemical B does not produce a higher frequency of reversion than the control and if this result is obtained for each type of mutation tested, then the results suggest that B is not a mutagen (Ames et al., 1973a; b).
Several different types of his mutants are used to test for different classes of mutagens. For example, frameshift mutagens will revert a frameshift mutation in his, etc.
The S. typhimurium his mutant strains used have three additional properties that make them more sensitive to mutagens (McCann et al., 1975). First, they have an rfa mutation that makes the outer membrane more permeable to large molecules. Second, they have a mutation in the uvrB gene to eliminate NER. Third, they carry the plasmid pKM 101
26 which increases error-prone PRR of DNA damage. Thus, reversion of his mutations in these strains provides a sensitive test for a broad spectrum of mutagens.
As indicated above, some chemicals (called pro-mutagens) are not mutagenic unless metabolized to more active derivatives. To test for such pro-mutagens, an extract of rat liver enzymes (S9 extract) is included in the reversion assay (McCann et al., 1975).
The bacterial reverse mutation test is commonly employed as an initial screen for genotoxic activity and, in particular, for the point mutation-inducing activity. An extensive database has demonstrated that many chemicals that are positive in this test also are genotoxic in other tests. There are examples of mutagenic agents that are not detected by this test; reasons for this shortcoming can be ascribed to the specific nature of the endpoint detected, differences in metabolic activation, or differences in bioavailability. In addition, the prokaryotic cells utilized in bacterial reverse mutation test differ from mammalian cells in such factors as uptake, metabolism, chromosome structure and DNA repair processes. Tests conducted in vitro generally require the use of an exogenous source of metabolic activation. In vitro metabolic activation systems cannot mimic entirely the mammalian in vivo conditions such as metabolized activation of some pro-mutagens. The test therefore does not provide direct information on the mutagenic and carcinogenic potency of a substance in mammals. For these cases, usually other in vitro mammalian cell tests should be performed using different cell types and different endpoints, i. e., gene mutation and chromosomal damage. However, it is still important to perform the bacterial reverse mutation test.
27 1. 3. 1. 2. In vitro mouse lymphoma thymidine kinase+/- gene mutation assay
An in vitro mammalian cell gene mutation test can be used to detect gene alterations induced by chemical substances. While there are a number of cell lines that can be used, the L5178Y TK+1--3. 7. 2C mouse lymphoma cell line using the thymidine kinase (TK) gene is the cell line and assay of choice. The mouse lymphoma assay
(MLA) was chosen because of a body of research indicating that many types of genetic alterations are detected. The assay detects mutations known to be important in the etiology of cancer and other human genetically mediated illnesses. There is evidence that the assay detects gene mutations (point mutations) and chromosomal events (deletions, translocations, mitotic recombination/gene conversion and aneuploidy) (Hozier, et al.,
1981; Moore, et al., 1985; Sawyer, et al., 1989; Applegate et al., 1990). The efficiency of detection of all of these mutational events is still under investigation.
There are some other in vitro mammalian gene mutation assays. For example, the assays using either Chinese hamster cell lines (CHO; Carver et al., 1983; Tindall et al.,
1986) or human lymphoblastoid cells (Crespi et al. 1985) have been reported. In these cell lines the most commonly used genetic endpoints measure mutation at either the hypoxanthine-guanine phosphoribosyl transferase (HPRT), a transgene of xanthine guanine phosphoribosyl transferase (XPRT), or TK. The TK, HPRT and XPRT mutation tests detect different spectra of genetic events. The autosomal location of TK allows for the detection of genetic events that are not detected at the HPRT locus on the X chromosome (Moore et al., 1989).
The various mutation assays are capable of detecting different spectra of genetic damage. Thus, it is not expected that a chemical will give uniformly positive or negative
28 results in the various assays. In particular, the bacterial Salmonella assay detects only point and other very small-scale gene mutations. Furthermore, the in vitro mammalian assays using the HPRT locus are unable to detect chemicals that do not induce point mutations (Moore et al., 1989).
1. 3. 1. 3. Mammalian erythrocyte micronucleus test
The purpose of the micronucleus test is to identify substances that cause cytogenetic damage that result in the formation of micronuclei containing lagging chromosome fragments or whole chromosomes. Micronuclei are cytoplasmic chromatin-containing bodies formed when acentric chromosome fragments or chromosomes lagging during anaphase and fail to become incorporated into daughter cell nuclei during cell division.
Because genetic damage that results in chromosome breaks, structurally abnormal chromosomes, or spindle abnormalities leads to micronucleus formation, the incidence of micronuclei serves as an index of these types of damage. It has been established that essentially all agents that cause double strand chromosome breaks induce micronuclei.
Since the detection of micronuclei is much faster and less technically demanding, and the micronuclei arise from two important types of genetic damage (clastogenesis and spindle disruption), the micronucleus assay has been widely used to screen for chemicals that cause these types of damage (Kirsch-Voiders et al., 1997)
The mammalian erythrocyte micronucleus assay is widely used. This in vivo micronucleus test is employed for the detection of damage induced by the test substance to the chromosomes or the mitotic apparatus of erythroblasts by analysis of erythrocytes sampled in bone marrow and/or peripheral blood cells of animals, usually rodents. When
29 a bone marrow erythroblast develops into a polychromatic erythrocyte, the main nucleus is extruded; micronuclei that have been formed may remain behind in the otherwise enucleated cytoplasm. Visualization of micronuclei is facilitated in these cells using specific staining techniques and by the occurrence of cells a main nucleus. An increase in the frequency of micronucleated polychromatic erythrocytes in treated animals is an indication of induced chromosome damage.
This mammalian in vivo micronucleus test is especially relevant to assessing mutagenic hazards in that it allows consideration of factors of in vivo metabolism, pharmacokinetics and DNA-repair processes, although these may vary among species, among tissues and among genetic endpoints. An in vivo assay is also useful for further investigation of a mutagenic effect detected by an in vitro system. However, if there is evidence that the test substance, or a reactive metabolite, will not reach the target tissue, it is not appropriate to use this test.
1.3.2. Genotoxicity tests that assess cellular responses to DNA damage
1.3.2.1. SOS Chromotest
In the early 1980's, the SOS Chromotest was described (Quillardet et al., 1982), in which a lacZ gene is placed downstream of the promoter of one of the SOS genes such as sfiA to allow a simple and direct colorimetric assay of the SOS response to DNA damage.
In view of its simplicity and its rapid response, the SOS Chromotest was proposed as a complementary or alternative test to the Ames test. The assay was shown to detect genotoxins inactive in the Ames test and to allow the identification of false positives
(Quillardet and Hofnung, 1993). Several other SOS genes were subsequently employed
30 to construct similar systems. In "umu-lacZ" test (Oda et al., 1985), the test organismS. typhimurium TA1535 has an excision repair deficiency (uvrB), increased membrane permeability (rfa ), and a natural deletion of the lac operon. The test allows chemicals that may be too toxic for the standard Ames test to be assayed. Vollmer et al. (1997) described a sensor system in which DNA damage-inducible promoters of recA, uvrA, alkA genes from E. coli were fused to luxABCDE of Vibrio fischeri. The regulation of recA and uvrA, which are a part of the bacterial SOS DNA repair system, is lexA dependent. Gene alkA encodes 3-mA-DNA glycosylase II. The alkA defective mutant strains are very sensitive to alkylating agents (Friedberg et al., 1995). The recA, uvrA, alkA fusion system exhibited the most prominent and sensitive responses to mitomycin C,
H202, MNNG, ethidium bromide (EtBr) or UV irradiation.
1.3.2.2. Common reporters in genotoxicity assays
To enable a cell to function as a biosensor, two elements are needed: a sensing element and a reporter element. The sensing element senses the presence of the target damaging agents, and turns on the reporter gene to emit a detectable signal. A good reporter should be readily detectable. Just as the specificity of the final construct depends upon the proper selection of the sensing promoter, the sensitivity and degree of resolution of the detection depend to a large extent upon the proper choice of the reporter. Several commonly used reporters are discussed below.
1.3.2.2.1. Bacterial B-galactosidase
p-galactosidase catalyzes the hydrolysis of colorless galactosides to yield colored
31 products. The enzyme is encoded by lacZ of E. coli and can be used in prokaryotic as well as in eukaryotic cells. The normal biological substrate for J3-galactosidase is lactose.
Under laboratory conditions, ortho-nitrophenyl-B-galactoside (ONPG) is usually used as a substrate. It is colorless and composed of galactose bonded to nitrophenol. J3- galactosidase will recognize this bond as a substrate and cleave this molecule to produce galactose and 0-nitrophenol (ONP) which is also colorless at neutral or acid pH, but in an alkaline solution it is bright yellow. The absorption of o-nitrophenolate can be measured at 420 nm. If ONPG is available in excess, then the amount of 0-nitrophenolate produced is proportional to the amount of enzyme (J3-galactosidase) present. The reaction is stopped when pH increases to 11 (with Na2C03), which inactivates J3- galactosidase. Since ONP is a product of J3-galactosidase activity, the spectrophotometric measurements may be used as an assay for the enzyme. Fluorescent assays have been developed for J3-galactosidase by using other substrates, such as 4-methyllumbelliferone, or fluorescein digalactoside.
1.3.2.2.2. Bacterialluciferases (LUC)
Luciferase is the protein that makes fireflies glow in the dark. It is found only in the gut of fireflies and several other marine organisms. Luciferases catalyze the obligatory aerobic oxidation of a reduced flavin mononucleotide and a long chain aldehyde to flavin mononucleotide and the corresponding carboxylic acid, with light emission at around 490 nm. The reaction is:
ATP + luciferin substrate+ 0 2 ~AMP+ oxyluciferin +LIGHT
32 Luciferase is encoded by luxA and luxB of the lux operon, and the synthesis enzymes for the aldehyde are encoded by luxCDE. In constructs where only luxAB is present, the aldehyde has to be added externally (Kohler et al., 2000). The great advantage of using luciferase as a reporter gene is that it is extremely sensitive. There is no background because luciferase is not found in mammalian cells. However, luciferase does require the luciferin substrate for activity. It also requires expensive equipment such as a luminometer or scintillation counter to detect the signal.
1.3.2.2.3. Green fluorescent protein (GFPl
Green fluorescent protein, GFP, is a spontaneously fluorescent protein isolated from coelenterates, such as the Pacific jellyfish, Aequoria victoria. Its role is to transduce, by energy transfer, the blue chemiluminescence of another protein, aequorin, into green fluorescent light. The molecular cloning of GFP eDNA and the demonstration that GFP can be expressed as a functional transgene have opened exciting new avenues of investigation in cell, developmental and molecular biology. Fluorescent GFP has been expressed in bacteria (Chalfie et al., 1994), yeast (Kahana et al., 1995), slime mold, plants, drosophila, zebrafish, and in mammalian cells (Prasher, 1995). It can function as a protein tag, as it tolerates N- and C-terminal fusion to a broad variety of proteins, many of which have been shown to retain native function (Cubitt et al., 1995). The advantage of GFP is that it requires no substrate; it constantly emits green light. The drawback is that the emission is not particularly strong. Therefore one must use a very strong promoter to increase expression of GFP so that one can see it within the cell. In addition, it is difficult to quantify.
33 1.3.2.2.4. Chloramphenicol acetyl transferase (CAT)
The oldest reporter assay relies upon the chloramphenicol acetyl transferase (CA1) gene. The CAT enzyme covalently modifies chloramphenicol by adding an acetyl group.
This reporter gene assay utilizes a proprietary colorimetric system. As the solution changes from yellow to red in wells containing CAT enzyme, the absorbance is read in a standard plate reader at 475-500 nm. The CAT enzyme activity is determined from the standard curve. The advantage for using the CAT gene is that it is not found in mammalian cells. Thus, there is no background. However, the CAT assay is not as sensitive as the luciferase assay. It is also rather expensive, time consuming, and radioactive isotopes are needed for some CAT systems.
The comparison of the above four reporters are summarized in Table 1-1. For a short-term genotoxicity test, reporters with the following characteristics are preferred: high sensitivity, wide dynamic ranges of response, ease of assay, inexpensive to assay and environmentally safe.
1.4. Development of a yeast-based RNRJ-lacZ genotoxicity testing system
Because of their simplicity, the Ames test and SOS Chromotests have become routine genotoxicity testing systems for environmental safety evaluation. Both of these systems are based on bacterial cells. Regarding the differences between eukaryotic and prokaryotic cells in metabolism, DNA damage responses and other physiological aspects, it is possible that an agent may be genotoxic for bacterial cells, but not for mammalian cells, and vice versa. The fact that some mammalian
34 Table 1-1: Comparison of commonly used f3-gal, CAT, LUC and GFP reporters
J3-gal LUC GFP CAT
Sensitivity 3 x 108 (ONPG) 6x16~=6xf67---~--- less sensitive 6x106'""6xtOW (molecules of 900,_, 1 (Fluorogenic than CAT, substrates) substrates) luciferase or f3- gal
Substrate required + + + Equipment Spectrometer* luminometer I fluorescent spectrometer w required scintillation counter microscope VI Degradation of + product
• Color filter assay. • f3-gal: f3-galactosidase; LUC: luciferase; GFP: green fluorescent protein; CAT: chloramphenicol acetyl transferase. genotoxic and carcinogenic agents cannot be detected in bacterial systems reflects these differences to some extent. 1,2-dimethylhydrazine is one example. As a well-known potent carcinogen, it cannot be detected as a bacterial mutagen in standard Ames test
(Lijinsky et al., 1985).
1.4.1. Advantages of yeast as host organism
The characteristics of the yeast S. cerevisiae have made it an ideal tool in scientific research and as such, it has been used extensively. There are essentially four major reasons to use yeast. First, although it is eukaryote, it shares the advantages of bacteria with regard to rapid growth, ease of manipulation and growth on a variety of carbon sources. Second, because it is eukaryotic organism, the metabolism and DNA damage induced responses likely resemble those of human responses. This is very important when using a biosensor to reveal potential hazards to other eukaryotes such as humans.
As was mentioned above, prokaryotic cells differ from eukaryotic cells in such factors as uptake, metabolism, chromosome structure and DNA repair processes. So the bacterial tests do not provide direct information on the mutagenic and carcinogenic potency of a substance in mammals. Third, among microorganisms, yeast is particularly robust with a wide physicochemical tolerance. These qualities are desirable in whole cell biosensors exposed to the real world. Yeast has a wide tolerance to pH, freezing temperature and osmolarity which ensures it can be used under field conditions. Finally, with regards to
S. cerevisiae, it is one of the best-understood and most-readily manipulated organisms. It is the first eukaryotic organism whose entire genome sequence was determined (Dujon et al., 1997; Bowman et al., 1997). The DNA damage response mechanism is well studied
36 compared to other eukaryotes. The wealth of knowledge with respect to yeast biology will enable us to modify the test yeast cells and alter the physiological and biological responses to environmental stress.
1.4.2. Rationale of the present RNR3-lacZ genotoxicity testing system
The rationale of RNR3-lacZ system is based on the induction of the DNA damage inducible gene RNR3 in response to DNA damaging agents. The model of RNR3 induction is reminiscent of the SOS response of E. coli. Hence, employment of the RNR3 gene promoter as a sensing element of genotoxicity testing system is actually comparable to that of SOS Chromotest. The maximum degree of RNR3 induction, according to previous data in our laboratory (Zhu and Xiao, 1998) and other laboratories (Ruby and
Szostak, 1985; Elledge and Davis, 1990; Yagle and McEntee, 1990), is much higher than that of other yeast genes examined. More importantly, it seems that RNR3 can respond to much broader spectrum of DNA damaging agents than bacterial cells.
In this study, a lacZ gene was placed downstream of the RNR3 promoter. When treated with DNA damaging agents, the RNR3 gene will be induced, initiating the expression of lacZ and produce p-galactosidase. By measuring the cellular level of induced p-galactosidase before and after treatment, it can easily be determined whether an agent is genotoxic.
1.5. Objectives of the present study
The objectives of this study were:
37 ( 1) to develop a eukaryotic genotoxicity testing system based on the budding yeast S.
cerevisiae;
(2) to test its responses to different DNA damaging agents;
(3) to improve the sensitivity of this system to different types of DNA damaging agents
by altering host yeast strains.
38 CHAPTER II: MATERIALS AND METHODS
2.1. Cell culture and manipulation
2.1.1. Yeast strains and cell culture
The yeast strains used in this study are listed in Table 2-1. Haploid S. cerevisiae strain DBY747 was used as the wild type host strain of the present RNR3-lacZ system.
YPD is a standard, complex medium composed of 1o/o Bacto-yeast extract, 2%
Bacto-peptone, 2% glucose, and 2% agar (if making YPD plates). Synthetic Dextrose minimal medium (SD medium) is used for selective growth of yeast auxotrophs. It contains 0. 67% yeast nitrogen base (without amino acids), 2% glucose, 2% agar (if making SD plates), supplemented with necessary amino acids and bases. If a necessary nutritional supplement (X) is absent in the media, it is denoted as SD-X. Yeast cells were grown at 30 °C either in rich YPD medium or in SD medium supplemented with appropriate amino acids and bases.
For long-term storage, yeast cells were grown on plates (rich or minimal media) at
30 °C. After 2-3 days growth, the yeast cells were removed from the plate with a sterile toothpick, and re-suspended into 1.0 ml of sterile 15% (v/v) glycerol. For yeast cells grown in liquid culture, after overnight growth in appropriate media, 0.7 ml of cells were added into 0.3 ml of 50% glycerol. The cells are then stored at -70 °C.
The cell titer is determined by the absorption at 600 nm on a Novaspecll visible spectrometer (Pharmacia).
39 Table 2-1. Yeast strains used in this study.
Strain Genotype Source
DBY747 MATa his3-11leu2-3, 112 trp1-289 ura3-52 Dr. D. Botstein
WXY9573 DBY747 with rad2t1::TRP1 Xiao et al., ( 1998)
WXY9376 DBY747 with rad6t1::LEU2 Xiao et al., (1996a)
WXY9326 DBY747 with rad18t1::TRP1 Xiao et al., (1996a)
WXY9221 DBY747 with rad50t1::HisG-URA3-hisG Xiao et al., (1996a) ~ 0 WXY9387 DBY747 with rad52t1::LEU2 Xiao et al., (1996a)
WXY814 DBY747 with apn1::HIS3 apn2::LEU2 Xiao et al., (200 1)
JC8901 DBY747 with mag1~::HisG-URA3-HisG Chen and Samson ( 1991)
WXY788 DBY747 with mag1t1::HisG apn1::HIS3 Xiao et al., (200 1)
apn2::LEU2 2.1.2. Yeast transformation
DBY747 cells carrying plasmids containing RNR3-lacZ, RNR2-lacZ, or MAGJ-lacZ were created by transforming yeast cells with pZZ2, pZZ18, or pWX1254, respectively.
Yeast cells were transformed using a method derived from Ito et al (1983) and Hill et al (1991 ). A 2 ml culture of S. cerevisiae was grown overnight at 30 °C in rich media.
The next day, the 2 ml culture was subcultured into 3 ml of fresh media, and allowed to grow at 30 °C until it reached a mid-logarithmic phase of growth. The yeast cells were collected by centrifugation, washed with a LiOAc solution (0.1 M LiOAc, 10 mM Tris
HCl (pH 8.0), 1 mM EDTA) and resuspended in 1 ml of LiOAc. For each transformation, 100 f.ll of cells from the 1ml suspension was mixed with 4 f.ll of ssDNA
(single stranded salmon sperm DNA) and 1-5 f.ll of transforming DNA in a 1.5 ml centrifuge tube. After a 5-minute incubation at room temperature, 280 f.ll of PEG4ooo solution (50% polyethylene glycol (MW=4000) in LiOAc solution) was added and the contents mixed by inverting 4-6 times. After the transformation mixture was incubated for 45 minutes at 30 °C, 39 f.ll of DMSO was added, followed by a 5-minute heat shock in
42 °C waterbath. After the heat shock treatment, the transformation mixture was washed with double distilled water (ddH20), and then resuspended in 0.1 ml of ddH20. The resuspended cells are plated onto the appropriate minimal media.
2.1.3. Yeast genomic and plasmid DNA isolation
The protocol for the extraction of yeast DNA was developed by Hoffman and
Winston ( 1987). Yeast cells from liquid culture were collected by centrifugation and resuspended in 200 f.ll of extraction buffer [2o/o TritonX-100, 1% SDS, 0.1 M NaCl, 1
41 mM EDTA, 0.01 M Tris HCl (pH8.0)]. 0.1 ml of phenol, 0.1 ml of chloroform and 0.3 g of acid-washed glass beads were added to the cell mixture. The tube was then vortexed for 3 minutes (2 minutes for isolating plasmid DNA) at top speed. After a 5-minute centrifugation, the top aqueous layer was transferred to a clean tube, and 2 volumes of
95% ethanol was added to precipitate the DNA. After 30 minutes at -20 °C, the DNA sample was centrifuged for 15 minutes, dried and resuspended in 200 J.il of ddH20 ( 10 mM Tris HCl, 1 mM EDTA, pH 8. 0), and then treated with 5 J.il ofRNase (10 mg/ml stock) at 37 °C for 10 minutes. The DNA was precipitated in 2 volumes of 100% ethanol with 8 J.il of 5 M NaCl and resuspended in 50 J.il of ddH20.
2.1.4. B-Galactosidase activity assay
For yeast cells carrying autonomously replicating plasmids, the ~-gal assay was performed as described (Xiao et al., 1993; Zhu and Xiao, 1998). Briefly, 0.5 ml of an overnight culture of yeast cells was used to inoculate 2.5 ml of fresh medium. After a 2- hour incubation, when cell density reached an OD600 of approximately 0.2-0.3, cells were treated with test chemicals or exposed to radiation and returned to incubation for another
4 hours or as specified. 1 ml of culture was used to determine the cell density at OD6oo.
The cells from the remaining 2 ml of culture were collected by centrifugation and used for the ~-gal assay. The cells were resuspended in 1 ml of buffer Z (60 mM Na2HP04•
1H20, 40 mM NaH2P04• H20, 10 mM KCl, 1 mM MgS04· 7H20, 40mM ~ mercaptoethanol, pH 7. 0), permeabilized by adding 50 J.il of 0.1% SDS and 50 J.il of chloroform and vortexed at top speed for 10 seconds. The reaction was initiated by adding 200 J.il of 4 mg/ml ONPG and incubating at 30 °C for 20 minutes. The reaction
42 was stopped by adding 500 J.ll of 1 M Na2C03. The tube was centrifuged and the OD420 value of the supernatant was measured to determine the p-gal activity using the following equation:
P-gal activity= 1000 (OD420nm)/ [reaction time (min) .culture volume (ml) • OD6oonm].
The P-gal activity is expressed in Miller units (Guarente, 1983). The value ofOD420 and
OD6oo were determined on a Novaspec II visible spectrometer from Pharmacia company.
The value of fold induction was calculated as a ratio of p-gal activity of the cells with and without treatment in the same experiment. The results were the average of at least three independent experiments with standard deviation calculated by Microsoft Excel2000.
For cells carrying an integrated RNR3-lacZ cassette in the genome, non-selective
YPD medium was used during incubation. 100 J.ll of overnight culture was inoculated into 2.9 ml of fresh YPD at a cell titer of approximately 0.1 at OD6oo. Cells were immediately treated with test chemical or exposed to radiation, and incubated for another
4 hours. The remaining steps were as described in the previous paragraph.
2.1.5. Chemicals and special media
All the agents and their mechanisms of function are listed in Table. 2-1. Genotoxic chemicals used in this study were MMS, EMS, SDMH, H202, 4-NQO, HU, 5- fluorouracil, phleomycin, MNNG and EtBr. Non-genotoxic chemicals were L canavanine and tetracycline (hydrochloride tetracycline). All the above chemicals were purchased from Sigma-Aldrich.
The 1.0 mg/ml MNNG stock solution was made in 100 mM acetate buffer (pH 5.0), aliquoted, frozen and thawed only once. Tetracycline was dissolved in ethanol
43 Table. 2-2: Agents used in the this study and their mechanisms of function Chemical Functional mechanism H202 Oxidative damage to DNA
4-NQO Cause bulky adducts~ UV radiation mimic, oxidative HU Inhibition of DNA replication (inhibitor of ribonucleotide reductase) MMS Alkylating agent EMS Alkylating agent EtBr Intercalating agent SDMH Alkylating agent MNNG Alkylating agent
+::-. Phleomycin Cleavage of DNA +::-. 5-Fluorouracil Base analog uv light Photoproducts in DNA, DNA cross-linking and strand breaks y ray Base, sugar damage and strand break Tetracycline Inhibit protein synthesis in prokaryotes (interacts with 30S subunit) Canavanine Non-specific inhibition of arginine decarboxylase activity
Note: 4-NQO: 4-nitroquioline; HU: hydroxyurea; MMS: methyl methanesulfonate; EMS; ethyl methanesulfonate; EtBr: ethidium bromide; SDMH: 1,2-dimethyl hydrazine; MNNG:N-methyl-N' -nitro-N-nitrosoguanidine; UV: ultraviolet light; Tetracycline: hydrochloride tetracycline and stored at -20 °C. All the other chemicals were freshly made in sterile ddH20.
5-Fluoro-orotic acid (FOA) is toxic to yeast cells with a functional URA3 (Boeke et al., 1987). The gene product of URA3 metabolizes FOA to a toxic intermediate. Fluoro orotic acid was purchased from US Biologicals (Swampscott, MA), and is stored as an aqueous solution at -20 °C. FOA plates were prepared as follows: A mixture of 1.34% bacto-yeast nitrogen base, 0.4% drop-out mix (-Ura), 4% glucose, 100 Jlg/ml uracil, 0.2%
5-FOA in 500 ml of ddH20 was filter sterilized. The above solution was combined with
500 ml of an autoclaved 4o/o bacto-agar, then pour into petri dishes.
2.1.6. Chemical and physical agents treatment
The chemical agent was added to the cells after 2 hours incubation. After treatment, cells were incubated for another 4 hours. Yeast cells were precipitated by centrifugation, washed twice with sterile ddH20, and resuspended in buffer Z for the P gal assay.
For UV treatment, cells were collected by centrifugation and resuspended in 200 Jll sterile ddH20, plated on YPD plate and exposed to 254 nm UV light in a UV cross-linker
2 (Fisher model FB-UVXL-1000) or UV lamp (UVP model UVGL-25 at 40 JlW/cm ) at given doses in the dark to prevent photoreactivation. The cells were washed, resuspended in 3 ml of liquid YPD/SD-Ura and incubated at 30 °C in the dark for another
4 hours prior to the P-gal assay.
For y ray irradiation, the cells were collected by centrifugation and resuspended in 1 ml of ddH20 in an eppendorf tube. The tubes containing yeast cells were exposed to a
45 6°Co y ray source, and the yeast cells were diluted into SD-Ura medium and incubated at
30 °C for another 4 hours prior to the P-gal assay.
2.1. 7. Cell killing
The cell killing test was used to determine the cell survival rates after treatment by chemical and physical agents. Chemical induced liquid killing was performed as previously described (Xiao et al., 1996). In order to maintain the transformed pZZ2 plasmids, cells were grown overnight in SD-Ura medium. 500 f.ll of overnight culture was subcultured into 5 ml of fresh medium the next morning and allowed to grow until mid
5 6 logarithmic growth was achieved. 1 ml of culture was removed, diluted 10 - and plated on SD-Ura plates. These cells represent the untreated control cells. At this time, chemical agent was added to the remaining culture, to the highest concentration as indicated in each experiment. After 4 hours of incubation, 1 ml of cells were removed, washed twice with sterile water, diluted to an appropriate concentration and plated in duplicate on SD-Ura plates. Plates were incubated for 3 days at 30 °C.
For UV killing, cells were plated at different dilutions and then exposed to 254 nm
UV light, either in a UV crosslinker (FB-UVXL-1000, FisherBiotech company) or with a
UV lamp (UVGL-58, UVP company) at specified doses. Cells were plated in duplicate on YPD to score cell survival, and the plates were incubated at 30 °C for 3 days.
Irradiation and the subsequent incubation of the cells were performed in the dark, to
prevent photoreactivation.
46 For y irradiation killing, cells were collected by centrifugation, resuspended in ddH20, and exposed to a 6°Co y -ray source in a centrifugation tube. The cells were plated on appropriate media. The plates were incubated for 3 days at 30 °C before counting the colonies.
2.2. Molecular biology techniques
2.2.1. Bacterial culture and storage
The E. coli strain commonly used for bacterial transformation was DH1 OB
(Gibico/BRL, Grand Island, NY USA). Transformed strains were stored on LB plates
(1% Bacto-tryptone, 0.5% Bacto-yeast extract, 0.5% NaCl and 1.2 % Agar) containing 50
J.Lg/ml of Ampicillin, since all of the vectors used contained the bla marker gene. For long-term storage of transformed cells, cells were grown overnight in 900 J.Ll of LB +
Amp; the following day 100 J.Ll ofDMSO was added and the cells were immediately placed in a -70 °C freezer.
2.2.2. Preparation of competent cells
For transformation by electroporation, E. coli cells were prepared as suggested in the BioRad E. coli Pulser manual. One litre of culture was incubated until an OD6oonm of
0.6 was reached. The culture was centrifuged at 3500 rpm in a Beckman GSA rotor and the pellet was resuspended in 500 ml of 1Oo/o sterile glycerol. The centrifugation was repeated 4 times, with each pellet resuspended in a reduced volume; the last pellet was
47 resuspended in 4 ml of cold, sterile 10% glycerol. The cells were ali quoted into 1.5 ml eppendorftubes to a volume of25 J..Ll, and were quickly placed in the -70 °C freezer for storage.
2.2.3. Bacterial transformation
All bacterial transformations in this study used the electroporation method. The
DNA to be transformed was added to competent E. coli cells. After a brief incubation on ice, the cell mixture was transferred to a chilled electroporation cuvette (BioRad), where the cells were exposed to a voltage of 1.8 kV (for cuvettes with 0.1 mm width) using the
E. coli Pulser (BioRad). 280 J..Ll of SOC medium was added to the cuvette after electroporation. The cell mixture was transferred to a 1.5 ml eppendorf tube for a 45- minute incubation at 37 °C. Following the incubation, the cells were plated on LB +
Amp plates and incubated at 3 7 °C overnight.
2.2.4. Plasmid DNA isolation (Mini-prep)
Plasmid amplification and isolation was performed according to the method of
Maniatis et al. (1982). Cells were inoculated into 1 ml of LB +Amp liquid media, and grown overnight at 37 °C. Cells were collected by centrifugation, the supernatant removed, and the pellet was resuspended in 350 J..Ll of STET (8% sucrose, 0.5o/o Triton
X-100, 50 mM EDTA pH 8.0, 10 mM Tris-HCI pH 8.0). After adding 20 J..Ll of lysozyme
( 10 mg/ml; Sigma, St Louis MI), the mixture was quickly put in a boiling water-bath for
30-40 seconds, followed by centrifugation for 10 minutes. The pellet was removed with
48 a toothpick, and 8 J.!l of 5 M NaCI and 2 volumes of 95% ethanol were added to precipitate the DNA. The DNA was then resuspended in ddH20 .
2.2.5. Agarose gel electrophoresis
For analysis of plasmid and genomic DNA, 0.8% agarose gel was used in this study. Gels were prepared in 1 x T AE buffer ( 40 mM Iris-acetate, 2 mM Na2EDTA).
To monitor the migration of the DNA, a sucrose dye (30o/o sucrose, 0.15% bromophenol blue, O.lM EDTA, pH 8.0) was added to the samples. Bacterial phage 'A DNA was digested with Hindiii, yielding DNA bands at 23.130, 9.416, 6.557, 4.361, 2.322, 2.027, and 0.564 kb. These bands were used to determine the size of the linear DNA of interest.
Electrophoresis was performed in 1 x T AE buffer using Fisher Biotech electrophoresis systems (Fisher Scientific, Pittsburgh, PA USA). The gel was stained in diluted EtBr solution (diluted from a 10 mg/ml stock solution) to visualize the DNA and photographed by UVP BioDoc-ltTM system for a permanent record.
2.2.6. Isolation of DNA fragment from agarose gel
The method of DNA fragment isolation from agarose gel was adapted from Wang and Rossman ( 1994). After enzyme digestion, the sample was electrophoresed through
0.8% agarose gel and stained with EtBr. The band of interest was identified and cut out of the gel under UV -illuminator. A 0.5 ml microcentrifuge tube was pierced at the bottom, and packed with chopped cheesecloth. The gel slice containing the DNA fragment was placed into the prepared tube, which was placed into another 1.5 ml tube,
49 left it in the -70 °C freezer for 30 min and spun for 10 min at top speed. The flow through was treated with the same volume of phenol/chloroform (1: 1) extraction and precipitated by ethanol.
2.2. 7. Ligation
To ligate a DNA fragment into a vector plasmid, approximately 0.5 J.lg of DNA insert was combined with 0.2 J.lg of plasmid vector. 1 J.ll ofT4 DNA ligase (Gibco/BRL) and 4 J.ll ofT4 DNA ligase buffer (5 X stock solution) were added to the DNA, resulting in a total volume of 20 J.ll. Ligation was performed overnight at 16 °C to promote the stable association of cohesive DNA ends.
2.2.8. Plasmids and plasmid construction
Plasmids pZZ2 and pZZ 18 (Zhou and Elledge, 1992) were obtained from Dr. S.
Elledge (Baylor College of Medicine, Houston, USA) and utilized to create the RNR3- lacZ and RNR2-lacZtesting systems, respectively. Plasmid pWX1254, constructed in our laboratory (Zhu and Xiao, 1998), was used to create the MAG 1-lacZ testing system.
Plasmid M4366 (Voth et al., 2001) was obtained from Dr. D. Stillman (University of
Utah, Salt Lake City, USA) and was used to integrate the RNR3-lacZ fragment into the
yeast genome. Table 2-2 summarizes all the plasmids used in this study.
In order to create a stable testing system, the RNR3-lacZ cassette was integrated
into the yeast host genome. Plasmid pZZ2 was digested with Nsil and EcoRI and a
fragment containing the 0.88 kb RNR3 promoter and the full length of lacZ was isolated
50 and inserted into Pasti and EcoRI digested plasmid pBluescript, then digested the constructed plasmid with BamHI to obtain RNR3-lacZ fragment and ligated the fragment into BamHI digested M4366 so that the RNR3-lacZ cassette together with a hisG-URA3- hisG cassette were flanked by 5' and 3' HO sequences with Notl sites on each side.
M4366 carrying the RNR3-lacZ fragment was digested with NotI and transformed into yeast cells. Through homologous recombination between the HO-hisG-URA3-hisG
RNR3-lacZ-HO cassette and the yeast genome, the RNR3-lacZ fragment was integrated into the host genome at the HO locus (Fig. 2-1 ). Positive clones were selected by assaying ~-gal activity and the hisG-URA3 pop-out derivatives were obtained by selection on a plate containing 5- FOA plates. The resulting strain was expected to contain only the RNR3-lacZ cassette plus one copy of hisG replacing the inactive HO gene.
2.2.9. Radioactive labeling of DNA fragments
The 0.9 kb HO fragment from plasmid M4366 and 2.0 kb of lacZ fragment from pZZ2 were used as RNR3 probe and lacZ probe (Fig. 2-1 ), respectively, for Southern analysis.
The DNA was labelled using the Random Primer Labeling kit from Gibco/BRL.
As stated in the manufacturer's instructions, 25 ng of DNA was placed in a boiling water bath to denature the DNA, followed by immediate storage on ice. The DNA was mixed with the random primers buffer mixture (0.67 M HEPES, 0.17 M Tris-HCl, 17 mM
MgCh, 33 mM 2-mercaptoethanol, 1.33 mg/ml BSA, 18 OD260 units/ml oligo
51 deoxyribonucleotide primers (hexamers), pH 6.8), dNTP's, 50J.!Ci of e2P]adCTP, and the Klenow fragment of E. coli DNA polymerase I, and incubated for more than 2 hours at 25 °C. Stop buffer was then added to stop the reaction; the DNA was precipitated by ethanol and re-suspended with ddH20.
Table 2-3: Plasmids used in this study, their genotypes and sources.
Plasmid Relevant characteristics Source/reference
pZZ2 YCp, URA3, RNR3-lacZ Zhou and Elledge ( 1992)
pZZ18 YEp, HIS3, RNR2-lacZ Zhou and Elledge ( 1992)
pWX1254 YEp, URA3, MAGJ-lacZ Zhu and Xiao (1998)
M4366 Yip, HO-hisG-URA3-hisG-poly-HO Voth et al., (200 1)
M4297 Yip, HO-poly-HO Voth et al., (200 1)
pBluescript SK(-) Phagemid vector Stratagene, CA, USA
52 Bglll
A HO-L I hisG I URA3 hisG jRNR31 lacZ I HO-R I X X < I HO-L I HO I HO-R I ? -3951 -2720 -1814 -717 0 ATG 1097 1199 1699 Bgl II Bglll Bglll
B t
~ HO-L I hisG I URA3 hisG IRNR31 lacZ HO-R ? + +
c t < HO-L hisG IRNR31 lacZ HO-R ? -3951 -2720 -1814 Bglll Bglll
D Probe: HO-L and lacZ
Fig. 2-1: Integration of the RNR3-lacZ cassette into the HO locus. A: RNR3-lacZ cassette was inserted into the plasmid M4366, flanked by a hisG-URA3-hisG cassette and a fragment from the right end of HO gene. After being linearized by Notl, the plasmid was transformed into yeast cells. Homologous recombination occurred between HO-L, HO-R and the chromosomal HO gene. B: The hisG URA3-hisG-RNR3-lacZ cassette was integrated into genomic HO locus. C: URA3 marker was popped out from the genome through homologous recombination between hisG.tandem repeats D: A 0.9 kb fragment from the left end of the HO gene and a 2.0 kb lacZ gene fragment were used as probes for Southern hybridization.
53 2.2.10. Southern hybridization
In order to confirm the genomic structure of the RNR3-lacZ integrated DBY747 strain, Southern hybridization was carried using a 0.9 kb HO fragment (Voth et al., 2001) and a 2.0 kb lacZ fragment as probes (Zhou and Elledge, 1992).
After digestion of the genomic DNA with Bgni, the DNA fragments were separated on a 0.8% agarose gel. The gel was then treated in solutions of 1) 0. 25M HCl for 10 minutes for depurination; 2) 0.4 M NaOH/0.6 M NaCl for 30 minutes for denaturation, and 3) 1.5 M NaCl/0.5 M Tris-HCl (pH 7.5) for 30 minutes for neutralization. The DNA was transferred from the gel to a nylon-based membrane (GeneScreen plus membrane,
DuPont) using 1Ox SSC (3M NaCl, 0.3 M tri-sodium citrate, pH 7 .0) overnight.
The following day, the membrane was placed into a hybridization bottle with 5 ml of pre-hybridization solution [2x SSC, 10% dextran sulphate, Sx Denhardt' s solution
(SOX stock: 10 g Fico11400, 10 g polyvinylpyrrolidone, 10 g bovine serum albumin; add ddHzO to 500 ml total volume), 50% formamide, and 1% SDS] and incubated at 42 °C in the hybridization oven for at least 2 hours. Before hybridization, 50 f..ll of boiled ssDNA
DNA and 60 J.d of probe were added to the prehybridization solution. The membrane was then incubated with the probe overnight at 42 °C. The membrane was washed twice for 5 minutes at room temperature in 2x SSC/0.1% SDS and washed twice at 65 °C in
0.2x SSC/0.1% SDS. The membrane was then placed with X-ray film at -70 °C and
developed.
54 CHAPTER III: CONSTRUCTION AND EVALUATION OF THE RNRJ-lacZ
SYSTEM
3.1. Introduction
One of the major concerns in the use of a bacterial short-term testing systems is that the genomic structure, genetic regulation and metabolism of bacteria are rather different from eukaryotic cells. Hence, in this study, a sensitive and stable genotoxicity testing system using S. cerevisiae was developed. The rationale is similar to that of SOS
Chromotest assay except that it is carried out in a yeast system. The RNR3 promoter is used as a sensing promoter to detect environmental damage to DNA; lacZ is fused to the
RNR3 promoter as a reporter. The fused RNR3-lacZ cassette can be introduced into the host cell either as a plasmid or integrated into the yeast chromosome. The /acZ gene was chosen as a reporter gene for the following reasons. First, it is very easily detected and does not require special expensive equipment. This is a critical point for future field use of this system. Second, as a common reporter gene to study gene expression, comparable data can be found in various publications, so that we can compare our results to other previous data and test their reliability. Third, there is no P-gal background since the lacZ gene is not found in yeast cells.
3.2. Results
3.2.1. RNR3 is a more suitable reporter than other DNA damage-inducible genes
An ideal sensor gene would respond to a relatively low dose of damage treatment and
have a high level of induction. Three such candidate genes, RNR2, RNR3, and MAGI
55 were compared for their inducibility by MMS in this study. As seen in Fig. 3-1, all three genes can be induced by MMS. However, the induction of RNR3-lacZ and RNR2-lacZ peaked at 0.02% whereas that of .MAGJ-lacZreached a maximum expression after 0.05%
MMS treatment. The maximum expression of RNR3-lacZ increased 53-fold while that of
RNR2-lacZ and .MAGJ-lacZ increased 19 and 6.6-fold, respectively. These results are consistent with previous studies utilizing both Northern hybridization and reporter gene analyses (Hurd et al., 1987; Elledge and Davis, 1989; Hurd and Roberts, 1989; Elledge and Davis, 1990; Zhou and Elledge, 1992; Xiao et al., 1993) as well as using different
DNA damaging agents including MMS, UV, y-ray and 4-NQO. A large number of yeast
DNA damage inducible genes whose induction profiles are similar to .MAG 1 have been analyzed, including DDIJ (Liu and Xiao, 1997), UBCJ3 (Brusky et al., 2000), PHRJ,
GGT2, RAD2 and RAD51 (unpublished work of Dr. Y Zhu), and their fold induction is comparable to or less than that of MAG 1. Therefore, it is concluded that RNR3-lacZ appears to be the best candidate among yeast DNA damage inducible genes studied to date.
3.2.2. Induction of the RNR3-lacZ system by different test agents
Genotoxic agents were selected to represent a broad range of DNA damaging agents that may or may not have a direct effect on mutagenesis (Table. 2-1), including
DNA alkylating agents (MMS, EMS, MNNG and SDMH), an oxidative agent (H202),
UV and a UV mimetic agent (4-NQO), ionizing radiation (y-ray), a base analog (5- fluorouracil) and an agent (phleomycin) that causes strand breaks. This test also included a ribonucleotide reductase inhibitor (HU) that interferes with DNA synthesis. The
56 c 0 40 ..(,) ,::s ,c 0 u. 20
0 0.04 0.08 0.12
Methyl methanesulfonate (%)
Fig. 3-1. Different expression level of RNR3-lacZ, RNR2-lacZ and MAGJ lacZ after MMS treatment. DBY747 cells carrying pZZ2 (RNR3-lacZ, c), pZZ18 (RNR2-lacZ, •) orpWX1255 (MAGJ-lacZ, o) were incubated in SD minimal medium and treated with various doses of MMS for 4 hours before P gal activity assay. The results are the average of three independent experiments and are expressed as fold of induction relative to their respective untreated cells.
57 mechanistic effects of these agents are well known (Friedberg et al., 1995). As seen in
Fig. 3-1 and 3-2, and summarized in Table 3-1, all11 tested genotoxic chemicals can induce the RNRJ-lacZ system to varying degrees. The sensitivity is comparable to or higher than the published SOS Chromotests values. For example, the threshold doses (at which the (3-gal activity increased by 2-fold) for MMS, EMS, HU and H20 2 in the sfiA lacZ system are about 0.10 mM, 10 mM, 10 mM and 1 mM, respectively (von der Hude et al., 1988), whereas the corresponding doses for the RNR3-lacZ test are 0.15 mM, 4 mM, 5 mM and 0.4 mM, respectively (Fig. 3-1 and 3-2). In this study, a well-known colon carcinogen, 1,2-dimethylhydrazine (SDMH) was studied. It was undetectable as a bacterial mutagen by the Ames test (McCann et al., 1975; Lijinsky et al., 1985) and SOS
Chromotests. Interestingly, the RNR3-lacZ system was induced by more than 8-fold after
0.1% SDMH treatment (Fig. 3-2-2). Hence, RNR3-lacZ induction appears to be a useful and complementary system to existing genotoxic and mutagenic tests.
Antibiotics such as tetracycline and canavanine, which kill yeast cells without damaging DNA, were also tested. Under our experimental conditions, at doses that kill as much as four-fifths or one-quarter of the yeast cells, respectively, tetracycline and canavanine were unable to induce RNRJ-lacZ expression (Fig. 3-2-3)
Table 3-1 summarizes various experimental results obtained from this study. It also attempts to compare the results of the RNRJ-lacZtest with the Ames test and SOS
Chromotests. Results of the Ames test and SOS Chromotests were obtained from the genotoxicity database: www. pasteur. fr/recherche/unites/pmtg/toxic/index. html.
The intercalating agent EtBr could not induce the RNR3-lacZ system (Fig. 3-3).
Since it also produced uncertain results in both Ames ( (Mamber et al., 1986) and SOS
58 Chromotest assay (Mamber et al., 1986; Quillardet and Hofnung, 1993), its capacity to cause DNA damage and mutation remains unclear.
3.2.3. Effects of incubation time on induction efficiency
In order to optimize test conditions for the RNR3-lacZ system, setting MMS as a reference compound, several parameters were altered to identify their effects on RNR3- lacZ expression.
The effect of varying duration of MMS treatment on RNR3-lacZ expression was determined. As seen in Fig. 3-4, MMS-induced J3-gal activity in RNR3-lacZ transformed cells increased with treatment time up to 4 hours and began to decrease after a 6- hour treatment. This result is consistent with the previous observation comparing a time course of MAGJ-lacZ induction with MAGI transcript by MMS (Xiao et al., 1993). In comparison, a recent report (Afanassiev et al., 2000) suggests that RNR2-GFP induction by MMS reached a maximum fluorescent signal after approximately 14-16 hours of incubation. Since the mRNA transcript level after MMS and other types of DNA damage treatment often reaches a peak after 30 minutes (Elledge and Davis, 1990), the delayed increase in J3-gal activity after DNA damage treatment probably represents the time required to produce the fusion protein and accumulate cellular ~-gal. The reduced ~-gal activity after 6 hours of treatment is either due to toxic effects that kill wild type cells, or due to turnover of the fusion transcript and/or protein.
3.2.4. The effects of cell growth stage on induction efficiency
The effect of cell growth stage on RNR3-lacZ induction by DNA damaging agents
59 c: T .Q 2 t> T :::l "0 .5 "0 (51 LL
0 8 0 2 0
HU (mM) uv EMS(%) y-ray 10
c: 0 T iS :::l ~ 5 "0 0 LL
0 0 0.2 0.4 0 0 0 0. 0
MNNG SDMH (%) 4-NQO Phleomycin
c: 0 iS :::l "03 .5 ::2 0 LL ~ - O+----r----1 12 0. 12 °
Canavanine 5-Fiuorouracil Tetracycline
Fig. 3-2. The induction of RNR3-lacZ by HU (hydroxyurea); UV (ultraviolet light); EMS (ethyl methanesulfonate), y ray, MNNG (N-methyl-N'-nitro-N-nitrosoguanidine); SDMH (1,2- dimethylhydrazine); 4-NQO (4-nitroquinoline), phleomycin, 5-fluorouracil, H202, tetracycline and canavanine. The DBY747 cells carrying pZZ2 (RNR3-lacZ) were incubated in SD minimal medium, treated with agents as indicated for a given dose and the incubation was continued for another four hours before ~-galactosidase activity assay. Each of the results was the average of three independent experiments. The fold of induction was calculated as a ratio of ~-galactosidase expressed in cells with and without treatment in the same experiment.
60 2~------
1.5- c 0 ;; u :s "C .E "C 0 LL
0.8
Ethidium bromide
Fig. 3-3. The response of RNR3-lacZ system to ethidium bromide. The DBY747 cells carrying pZZ2 (RNR3-lacZ) were incubated in SD-Ura medium, treated with ethidium bromide at doses and incubate for another four hours before assaying the p-galactosidase activity. The result was the average of three independent experiments. The fold of induction was calculated as a ratio of P-galactosidase of cells with and without treatment in the same experiment.
61 Table 3-1: Summary of the RNR3-lacZ test and comparison with other test results
Agents Basal level a Highest Fold Optimal Survival sos Ames test
(Miller unit) Inductiona induction doseb (%) teste MMS 0.96±0.12 51.78±7. 08 53.9 0.02% 19.6 + + yray 1.18±0.30 31.03±2. 48 26.3 30 K rad 45.5 N/Ad NIA EMS 0.59±0.06 13.16±1. 56 22.3 0.5% 42.1 + + 2 uv light 1.09±0.07 16.80±2. 51 15.4 50 J/m 84.5 + NIA HU 1.02±0.08 12.40±0. 15 12.2 75mM 28.8 + + SDMH 0.69±0.08 5.72±0. 37 8.3 0.1% 46.7 - +e
MNNG 1.12±0.09 7.85±0. 40 7.0 5 ~g/ml 80.1 + + 0\ N 4-NQO 0.93±0.05 7.76±0. 32 8.3 0.8 ~g/ml 27.1 + + Phleomycin 0.81±0.07 4.99±0. 76 6.1 0.15 mg/ml 49.7 + NIA H202 0.96±0.12 4.48±0. 75 4.7 0.6mM 63.2 +
5-Fluorouracil 1.21±0.05 4.69±0. 95 3.9 0.1 ~g/ml 52.5 +I- +/?f
Tetracycline 1.03±0.16 1.34±0. 04 1.3 100 ~g/ml 21.2 - -I? Canavanine 0.77±0.18 <1.0 100 Jlg/ml 54.9 NIA NIA
EtBr 0.59±0.08 0.60±0.17 1.0 0.04 ~g/ml 81.0% +/- +/- Note: 4-NQO: 4-nitroquioline; HU: hydroxyurea; MMS: methyl methanesulfonate; EMS; ethyl methanesulfonate; EtBr: ethidium bromide; SDMH: 1,2-dimethyl hydrazine; MNNG:N-methyl-N' -nitro-N-nitrosoguanidine; UV: ultraviolet light; Tetracycline: hydrochloride tetracycline
"+"indicates positive results; "-" inducates negative results
a Average ~-gal activity of untreated and treated samples at the highest induction level within experimental conditions, presented as Miller unit. All results are based on at least three independent experiments with standard deviation.
b Dose of treatment at which RNR3-lacZ reached highest induction, or the highest dose of treatment, within experimental limits.
c Results of SOS Chromostest assay and Ames test were obtained from database www. pasteur.fr/recherche/unites/pmtg/toxic/index. html
d Not available.
0\ w e Positive results were obtained only in DNA repair alkyltransferase deficient strains (Xiao et al., 1996).
f Unclear interpretation was also explored. This study was focused on log phase cells, since it was anticipated that most DNA damaging agents attack replicating DNA during cell division and that RNRJ
DNA damage induction requires cell cycle checkpoints (Zhou and Elledge, 1993; Navas et al., 1995) which function during cell division. The cell titer prior to the P-gal assay ranging
7 7 from lx 10 to 3x10 cells/ml (OD600 = 0.2-0.7) affects p-gal specific activity up to 12-fold; however, the relative induction remained unaltered (Fig. 3-5). In contrast, the induction of
RNRJ-lacZ in stationary phase cells reached a maximum of 2.5 fold after treatment with
0.0025% MMS. Hence, the RNRJ-lacZ test is relatively independent of cell titer as long as cells are in the log phase. Results obtained in this study were typically performed when
7 asynchronized cells reached 1.5-1.8 x 10 cells/ml (OD6o0=0.4).
3.2.5. Creation and evaluation of a stable RNR3-IacZ testing system
The RNRJ-lacZ reporter gene used in the above studies was carried in a centromere based YCp plasmid which is a single copy plasmid with a plasmid loss rate of 10-3 per cell per generation. The experiments were conducted in selective medium to ensure that only cells containing the plasmid were able to grow and divide. In order to increase the stability of the testing system and to make it more user-friendly, the RNR3-lacZ reporter was integrated into the host genome at the HO locus.
3.2.5.1. Integration of RNR3-lacZ cassette into the yeast genome
The HO gene encodes a site-specific endonuclease; however, HO in most laboratory strains, including the strains used in this study, is inactive (Baganz et al.,
1997). The RNR3-lacZ cassette was delivered to the HO locus using a recently
64 c 60 0 ;: CJ :::s "'C c 40 "'C 0 LL 20
0 0.01 0.02 0.03
Methyl methanesulfonate (%)
Fig. 3-4. Effects of incubation time on the induction efficiency of RNR3- lacZ expression. The pZZ2 transformants were incubated in SD minimal medium, treated with MMS at the given final concentration, and incubated for 3 (o), 4 (•), 6 (o), 8 (•) and 10 (A) hours before p-gal activity assay. Each of the results was the average of three independent experiments. The fold of induction was calculated as a ratio of P galactosidase of cells with and without treatment in the same experiment.
65 0 0.02 0.04
Fig. 3-5: Effects of cell growth stage on induction efficiency. The pZZ2 transformants were grown in SD minimal medium to OD6oo = 0.06 (o ), 0.13 (•) and 0.21 (c), treated with MMS at the given final concentration, and incubated for 4 hours before p-gal activity assay. Cell titer at the 7 time of P-gal assay for the above samples were approximately 1 x 10 , 2 x 10 7 and 3 x 10 7 cells/ml, respectively. Each of the results was the average of three independent experiments.
66 developed integration system (V oth et al., 2001) such that, after initial integration and subsequent selectable pop-out through homologous recombination, only the RNR3-lacZ cassette plus one copy of hisG were integrated to replace the inactive host HO sequence, resulting in a single-copy reporter gene in the haploid host chromosome. Hence, it is expected to be as stable as any other yeast nuclear gene. The genomic structure (Fig. 2-1) was confirmed by Southern hybridization (Fig. 3-6).
3.2.5.2: Evaluation of the integrated RNR3-lacZ system
The resulting yeast strain was cultured in non-selective rich medium and the p-gal activity was compared to the same yeast strain carrying a plasmid-based reporter gene grown in the selective medium. As shown in Fig. 3-7, the induction profiles of three representative testing compounds (MMS, HU and 4-NQO) are indistinguishable between the integrant and plasmid-based systems. The results indicate that the RNR3-lacZ integrant strain provides a suitable genotoxic testing system complementary to the existing microbial testing systems.
67 1 2 3 4
23130 9416 6554 4361
2322 2027
564
Probe--+- HO lacZ
Fig. 3-6. The genomic structure of integrated RNR3-lacZ system was confirmed by Southern hybridization. Lane 1, 3: genomic DNA from DBY747 wild type strain; Lane 2, 4: genomic DNA from DBY747 integrated RNR3-lacZ cassette. The lacZ fragment was detected in the integrated cell (lane 4) but not in wild type DBY747 (lane 3). Because the integration of the RNR3-lacZ cassette into the HO locus, the fragment size increased from 3 .2kb to 8kb when hybridized with HO probe (lanes 2 and 4).
68 60 15 - 10 - c lA B lc 0 A I I I ;:: ,_=(,) .5 "C 0 u.
0-t;f 0 I I I 0 0.015 0.03 0 75 150 0 0.5
MMS {0/o) HU (mM) 4-NQO (JJ.g/ml)
0\ \.0
Fig. 3-7: Comparison of RNR3-lacZ induction between the plasmid-borne and the integrated RNR3- lacZ reporter. DBY747 cells carrying pZZ2 (w) were incubated in SD minimal medium to an OD600 of 0.2-0.3, and those containing a RNR3-lacZ integrant at the HO locus (u) were incubated in YPD to an OD600 ofO.l. Cells were then treated with MMS (methyl methanesulfonate, A), HU (hydroxyurea, B) or 4-NQO (4-nitroquioline, C) at the given concentration with further incubation of 4 hours before p-gal activity assay. All the results are an average of at least three independent experiments and are expressed as fold induction relative to untreated cells. The fold of induction was calculated as a ratio of b-galactosidase of the cells with and without treatment in the same experiment. CHAPTER IV: ENHANCING THE SENSITIVITY OF RNR3-lacZ SYSTEM BY
GENETIC MODIFICATION OF THE HOST STRAIN
4.1. Introduction
The RNR3-lacZ genotoxicity testing system was developed as previously described
(see Chapter Ill). It is a simple testing system with characteristics of high sensitivity, ease of assay, and time and cost saving. A variety of physical and chemical agents were tested in this system (Jia et al., 2002). The results correlate with the Ames test very well.
Based on the comparison of minimum response dose with SOS Chromotest on several chemical agents, the sensitivity of the RNR3-lacZ system is comparable to or even higher than the SOS Chromotest (Jia et al., 2002).
There have been a variety of attempts to enhance the sensitivity of the Ames test. It has been shown that by adding S9 rat liver extract into the system, some carcinogens can be activated to mutagens and readily detected (Ames et al., 1973a). The rfa mutation in the strains leads to a defective lipolysaccharide (LPS) layer that coats the bacterial surface and makes the cells more permeable to bulky chemicals (Ames et al., 1973b).
Modifying the DNA repair system is another choice to enhance the sensitivity of the Ames test. It is found that introducing an R factor (pKM101) into Ames strains can enhance chemical and UV-induced mutagenesis and make the test more sensitive
(McCann et al., 1975; Walker and Dobson, 1979). Based on this theory, many R factor transformed strains such as TA100, TA98 (Ames et al., 1975), TA97 (Levin et al., 1982),
TA102 and TA104 (Levin et al., 1982) were employed in the Ames test. Another attempt was to delete the uvrB gene, which is involved in nucleotide excision repair in E. coli. In the uvrB mutant strain, the incidence of mutagenesis was enhanced and therefore lowered
70 the threshold of the Ames test (Moreau et al., 1976). Similar efforts were also reported with the SOS Chromotest. It was found that the introduction of a dam-3 mutation into a
PQ37 derivative strain used in the SOS Chromotest enhanced the sensitivity of the system
(Quillardet and Hofnung, 1987). The author provided evidence that the increase in SOS inducibility due to the dam-3 mutation is specific for compounds causing DNA mismatches. Previous research in our laboratory (Xiao et al., 1996) also demonstrated that SDMH could be detected as a bacterial mutagen if Ames test strains were defective in the DNA repair 0 6 -methylguanine methyl transferase activity whereas the standard
Ames test failed to detect it as bacterial mutagen (Lijinsky et al., 1985). The above results suggest that, as with the Ames test and SOS Chromotest, the sensitivity of eukaryotic genotoxicity testing systems may be further improved by inactivating certain
DNA repair pathways. The fact that deletion of RAD16 in yeast cells enhances the sensitivity of a number of yeast DNA damage inducible genes to DNA damage treatment
(Kiser and Weinert, 1996) supports this hypothesis. Most DNA repair pathways dealing with different types of DNA lesions have been well characterized in budding yeast and mutations in each of these pathways have been created in our laboratory. This hypothesis was systematically tested in the present study.
Unlike prokaryotic cells, the regulation of the DNA repair network seems much more complicated. Since the early 1970's, an extensive collection of yeast mutants (rad) that are sensitive to UV radiation at 254 nm and/or to ionizing radiations has been established (Cox and Parry, 1968; Game and Mortimer, 1974). According to their responses to UV radiation and ionizing irradiations, these mutants were placed into three epistasis groups (Game and Cox, 1971 ). It became clear that these groups represent three distinct repair pathways, namely the RAD3 nucleotide excision repair pathway, the
71 RAD52 DNA recombinational repair pathway and the RAD6 post-replication repair and mutagenesis pathway (Friedberg et al., 1995). In addition to the above rad genes, another group of genes were found to be mainly involved in the repair of damaged bases and functioned in the base excision repair pathway.
In this study, mutant strains (Table 2-1) defective in DNA repair genes from different DNA repair pathways were employed as the host strain of the RNR3-lacZ system. The expression level of RNR3 was measured and compared with that of the wild type DBY747. Several mutant strains have been found to enhance the inducibility of the
RNR3-lacZ reporter gene.
4.2. RESULTS
In this study, isogenic deletion mutants ofDBY747 from different repair pathways were used as host strains of the RNR3-lacZ system. The peak induction of RNR3 as well as the dose level at which the induction was obtained were compared with the original
RNR3-lacZ system in the wild-type strain DBY74 7.
4.2.1. The effects of base excision repair mutation on RNR3-IacZ induction
All BER reactions are initiated by the action of a specific class of DNA enzymes called DNA glycosylases. The glycosylase recognizes and binds to the damaged site, then mediates the cleavage of the damaged base from the sugar backbone. The resulting abasic site is incised exclusively by AP endonucleases (Wallace, 1994; Sancar, 1994;
Seeberg, 1995). BER mediated by AP endonucleases is the primary defense against AP sites and 3'-blocked SSBs (Lindahl and Wood, 1999; Hoeijmakers, 2001)
72 MAGI encodes a 3-methyladenine (3MeA) DNA glycosylase (Chen et al., 1989), the first enzyme in a multistep BER pathway for the removal of lethal lesions such as
3MeA, and protects yeast cells from killing by DNA alkylating agents such as MMS
(Sakumi and Sekiguchi, 1990).
Two endonucleases were found inS. cerevisiae, namely Apn1 and Apn2, encoded by APNJ and APN2, respectively. Apn1 and Apn2 catalyze the hydrolytic cleavage of the phosphodiester backbone at the 5' side of an AP site, yielding an SSB with a 3'-0H group (Demple and Harrison, 1994; Unk et al., 2000). The apnl mutants were moderately sensitive to the killing action of alkylating agents such as MMS and exhibited enhanced spontaneous mutation rates (Ramotar et al., 1991 ). Deletion of APN2 alone did not produce any noticeable phenotypic alterations; however, Apn2 provides a strong backup for the Apn1 AP endonuclease activity. Cells lacking both AP endoculeases are remarkably sensitive to the cytotoxic effects of alkylating agents, display an elevated spontaneous mutation rate and lose the ability to repair AP sites in genomic DNA
(Johnson et al., 1998; Bennett, 1999). In this study, the apnl apn2 double mutant strain was used to test the effect of their deletion on the RNR3 induction. Since it was also reported that the mutator phenotype of apnl is profoundly affected by 3MeA levels (Xiao and Samson, 1993; Glassner et al., 1998; Xiao et al., 2001), the magi apnl apn2 triple deletion mutant strain was also employed to determine whether RNR3 induction can be increased in this genetic background.
With the mag1 null mutant strain, the expression of RNR3 after MMS treatment was increased (Fig. 4-1 ). In particular, the maximum induction of RNR3-lacZ increased from 48-fold in wild type cells to 59-fold in the magi null mutant. More importantly, the minimum and maximum induction doses decreased dramatically in the mag1 mutant.
73 Hence, with MMS doses up to 0.01 %, the magi mutant strain displays a 3-4 fold enhancement of induction (Fig. 4-1 ). This phenomenon appears to be true for other DNA alkylating agents, since the magi mutant shows an enhanced sensitivity to another alkylating agent, EMS. In this case, although the maximum fold induction is similar between wild type and mutant cells, the mag1 mutant reaches the maximum at 0.2%
EMS, instead of 0.5o/o in wild type cells. A 4-5 fold enhancement of RNR3-lacZ induction at low doses of EMS by MAG 1 deletion was observed and the response is in the linear range (Fig. 4-2). In contrast, deletion of MAGI does not affect the RNR3-lacZ induction profile by UV treatment (Fig. 4-3), and actually decreases the RNR3-lacZ response to y-ray treatment at high doses (Fig. 4-4 ). These results indicate that the enhancement of RNR3-lacZ sensitivity by MAGI deletion is probably specific to DNA alkylating agents and limited to lesions repaired by the Mag1 DNA glycosylase.
The enhanced sensitivity of the RNR3-lacZ genotoxic testing system to DNA alkylating agents in a MAGI deletion strain seems to suggest that it is due to the increased sensitivity of magi cells to killing by MMS. Deletion of both AP endonuclease genes in yeast results in extreme sensitivity to killing by MMS (Johnson et al., 1998; Bennett, 1999) and other base-damaging agents (Bennett, 1999). RNR3-lacZ induction was tested by
MMS in the apnl apn2 double mutant and surprisingly found that it was actually decreased
(Fig. 4-1). Deletion of the MAGI gene in the AP endonuclease deficient cells partially rescues the severe sensitivity to killing by MMS (Xiao et al., 2001 ); however, the mag1 apnl apn2 triple mutant almost completely lost the RNR3 induction (Fig. 4-1 ).
74 75-~------~
T
c: 0 ~ (,) ::s "C c: "C 0 LL
0 0.02 0.04
Methyl methanesulfonate (%)
Fig. 4-1. Comparison ofMMS-induced RNR-lacZ expression in the magi null mutant (•), the apn apn2 mutant (•) and maglapnlapn2 mutant (6) with that in the wild type DBY747 strain (c). Yeast cells carrying pZZ2 (RNR3-lacZ) were incubated in SD-Ura medium, treated with MMS as indicated doses and incubated for an additional four hours before assaying p-galactosidase activity. The result was the average of three independent experiments. The fold of induction was calculated as a ratio of P-galactosidase expressed in cells with and without treatment in the same experiment.
75 30------~
c 0 ;; u :::J "C c "C 0 LL
0 0.5
Ethyl methanesulfonate (%)
Fig. 4-2. Comparison of RNR3-lacZ induction in the mag] null mutant (c) and in the wild type DBY747 (•) after EMS treatment. RNR3-lacZ induction can be detected at much lower EMS dose in mag] mutant than in DBY747. Yeast cells carrying pZZ2 (RNR3-lacZ) were incubated in SD-Ura medium, treated with EMS as indicated dose and incubated for an additional four hours before assaying the ~-galactosidase activity. The result was the average of three independent experiments. The fold of induction was calculated as a ratio of P galactosidase expressed in cells with and without treatment in the same experiment.
76 c 0 ;; (,) ::s "'Cc "'0 0 LL
0 40 80
2 Ultraviolet Light (J/m )
Fig. 4-3. UV-induced RNR3-lacZ expression in wild type DBY747 (•) and the magi null mutant (c). The two strains show no significant difference in the response. The yeast cells carrying pZZ2 (RNR3-lacZ) were incubated in SD Ura medium, treated with UV as indicated dose and incubated for additional four hours before assaying p-galactosidase activity. The result was an average of three independent experiments. The fold of induction was calculated as a ratio of p-galactosidase expressed in cells with and without treatment in the same experiment.
77 40------~
r::: 0 l ..(.) ,:s ,·-r::: 0 u.
80
'Y ray (krad)
Fig. 4-4. Comparison of RNR3-lacZ induction in the magl deletion mutant (•), the rad2 deletion mutant (•) and the wild type DBY747 (c) after y-ray treatment. Yeast cells carrying pZZ2 (RNR3-lacZ) were incubated in SD-Ura medium, treated with agents as indicated dose and the incubated for additional four hours before assaying p-galactosidase activity. The result was an average of three independent experiments. The fold of induction was calculated as a ratio of P-galactosidase expressed in cells with and without treatment in the same experiment.
78 4.2.2. Deletion of nucleotide excision repair gene RAD2 enhances the induction of
RNR3-lacZ to UV radiation
NER consists of the process of damage recognition, dual incision, release of an oligonucleotide fragment carrying the damage, re-synthesis of the gap, and finally ligation of the free ends. The incision step ofNER is mainly completed by the RAD3 epistasis group. This group was further divided into two classes: Class 1 consists of
RADJ, RAD2, RAD3, RAD4, RADiO, RAD14 and RAD25; Class 2 contains RAD7,
RAD16, RAD23 and MMS19. It is suggested that Class 1 genes are essential for all the steps leading to the incision of damaged DNA, while Class 2 genes only affect the proficiency of the reaction. Indeed, mutations in Class 1 genes confer a much higher degree of sensitivity to UV light and other DNA damaging agents and cause a high level of defect in the incision of UV damaged DNA and crosslinked DNA (Prakash and
Prakash, 2000).
Based on the essential roles of Class 1 genes in the NER pathway, one of them,
RAD2, was selected in this study to determine how its deletion mutation affects the
RNR3-lacZ induction, in order to assess the potential application to enhance the
2 sensitivity of the RNR3-lacZ system. Rad2 protein has a Mg + dependent endonuclease activity that is specific for single-stranded DNA (Habraken et al., 1993) and cleaves at the 3 '-side of the lesion (Habraken et al., 1995). Furthermore, RAD2 expression is UV inducible at the transcriptional level (Siede and Fridberg, 1992).
The induction of RNR3-lacZ after UV treatment was less than 20-fold in wild type
79 cells (Jia et al., 2002; Fig. 4-5). It was surprising to find that the peak level reached more than 40-fold in the rad2 null mutant (Fig. 4-5). More importantly, the maximum induction was reached at 10 J/m2 in the rad2 mutant compared to 50 J/m2 in the wild type cells (Fig. 4-5). A UV mimetic agent 4-NQO was tested to see if inactivation ofNER is able to help detection of genotoxic chemicals. Indeed, deletion of RAD2 enhances the induction of RNR3-lacZ by about 3 fold in the dose range examined (Fig. 4-6). It was also reported that NER participates in the repair of DNA methylation damage (Xiao et al.,
1996; 1998). Hence, the effect of deleting RAD2 on the MMS-induced RNR3-lacZ expression was examined. It was interesting to notice that at low MMS doses, the rad2 mutant displayed an enhanced sensitivity. However, at doses higher than 0.1% MMS,
RNR3-lacZ induction was compromised in the rad2 mutant (Fig. 4-7). Deletion of RAD2 also compromised RNR3-lacZ induction by y-ray treatment at high doses, although no significant differences between the rad2 mutant and wild type cells was observed at low doses (Fig. 4-4 ).
4.2.3. Effects of recombination repair mutations on the induction of RNR3-IacZ
Recombination repair appears to be much more complicated than other DNA repair pathways. The present understanding about the mechanism of this pathway especially regarding the homologous recombination has lagged behind that of other DNA metabolic processes. DSBs are induced by a variety of DNA-damaging agents, including ionizing radiations like y-rays and X-rays, as well as radiomimetic chemicals such as MMS.
The DSBs may also arise "spontaneously" from replication past SSBs, processing
80 50------~
c: l 0 ;; CJ ,::l ,c: 0 u.
0 40 80
2 Ultraviolet light (J/m )
Fig. 4-5. Comparison of RNR3-lacZ induction level in wild type DBY747 (c) and in rad2 mutants (•) after UV treatment. The peak level in the rad2 deletion mutant 2 reached more than 40 fold at 10 J/m , while the peak level in DBY747 was less than 2 20-fold, which was reached at a much higher dose level of 45 J/m • The yeast cells carrying pZZ2 (RNR3-lacZ) were incubated in SD-Ura medium, treated with UV as indicated dose and incubated for additional four hours before assaying ~ galactosidase activity. The result was an average of three independent experiments. The fold of induction was calculated as a ratio of ~-galactosidase expressed in cells with and without treatment in the same experiment.
81 30------~
c: 0 +:; T u ::::s "C c: "C 0 LL
0 0.4 0.8 1.2
4-nitroquinoline (J.lg/ml)
Fig. 4-6. Comparison of RNR3-lacZ induction in wild type DBY747 (c) and its rad2 null mutant (•) after 4-NQO treatments. The fold induction in the rad2 mutant is higher than that in DBY747 and reached >20 fold, whereas that in DBY747 was less than 10-fold. The yeast cells carrying pZZ2 (RNR3-lacZ) were incubated in SD-Ura medium, treated with 4-NQO as indicated dose and incubated for additional four hours before assaying ~-galactosidase activity. The result was an average of three independent experiments. The fold of induction was calculated as a ratio of ~-galactosidase expressed in cells with and without treatment in the same experiment.
82 60
c T 0 +i (,) ::J 40 "Cc "C 0 LL
0~------~------~ 0 0.02 0.04
Methyl methanesulfonate {0/o)
Fig. 4-7. Comparison of RNR3 induction level in DBY747 (c) and rad2 null mutant strain (•) after MMS treatment. At lower doses, the rad2 mutant displayed an enhanced sensitivity; at doses higher than 0.1% MMS, the RNR3-lacZ induction was compromised in the rad2 mutant. The yeast cells carrying pZZ2 (RNR3-lacZ) were incubated in SD-Ura medium, treated with MMS as indicated dose and incubated for additional four hours before assaying ~-galactosidase activity. The result was an average of three independent experiments. The fold of induction was calculated as a ratio of ~-galactosidase expressed in cells with and without treatment in the same experiment.
83 of endogenous damage and endonuclease activities (Friedberg et al., 1995).
RAD52 epistasis group are involved in the repair ofDSBs inS. cerevisiae.
Although these genes share the property of conferring resistance to ionizing radiation, their mutants display considerable variation in assays for DSB repair and recombination.
RAD50, XRS2, and MREJJ form a subgroup with similar properties. Physical interactions among their gene products have been detected by the two-hybrid system (Johzuka and
Ogawa, 1995; Chamankhah and Xiao, 1999). It has been suggested that the complex is nucleolytic and plays a role in the initiation of homologous recombination as well as nonhomologous end rejoining (Johzuka and Ogawa, 1995; Moore and Haber, 1996).
Rad52 is known to bind to the ends of DNA molecules (Dyck et al., 1999) to promote
DNA strand annealing between single-stranded oligonucleotides (Mortensen et al., 1996;
Sugiyama et al., 1998), and to potentiate the strand-exchange activity of Rad51
(Shinohara and Ogawa, 1995; Sung, 1997; New et al., 1998;). Its mutants produce the
most severe defects in DSB repair and recombination (Friedberg et al., 1995).
In this study, both rad50 and rad52 null mutant strains were employed. The
induction of RNR3-lacZ after treatment of y-ray was dramatically reduced in both rad50
and rad52 cells compared with the wild type cells (Fig. 4-8). It is noticed that the basal
levels of ~-gal activity in rad50 and rad52 mutants are approximately 4.0 and 7.0,
respectively, while it is less than 1.0 in wild type cells, which may partially explain the
compromised fold induction in the recombination repair deficient cells. Similarly, MMS
induced RNR3-lacZ expression was also compromised in the rad52 mutant (Fig. 4-9) to a
level comparable to the y-ray treatment (Fig. 4-8).
84 30- l c 0 ;; (,) ::l "C c "C 0 LL
0 25 50 75
y ray (krad)
Fig. 4-8. Comparison of RNR3 induction level in DBY747 (c), rad50 deletion mutant strain ( •) and rad5 2 deletion mutant (o) after y-ray treatments. Both of the two mutant strains reduced the induction of RNR3 dramatically comparing to DBY747. The yeast cells carrying pZZ2 (RNR3-lacZ) were incubated in SD-Ura medium, treated withy ray as indicated dose and incubated for additional four hours before assaying p-galactosidase activity. The result was an average of three independent experiments. The fold of induction was calculated as a ratio of P galactosidase expressed in cells with and without treatment in the same experiment.
85 c 40 0 ;; (J ::s "'C c "'C 0 20 LL
0 0.02 0.04 0.06
Methyl methanesulfonate (o/o)
Fig. 4-9. Comparison of RNR3-lacZ induction in the wild type DBY747 (c) and rad52 mutant strain(•) after treatment with MMS. Yeast cells carrying pZZ2 (RNR3-lacZ) were incubated in SD-Ura medium, treated with MMS as indicated and the incubated for additional four hours before assaying p-galactosidase activity. The result was an average of three independent experiments. The fold of induction was calculated as a ratio of p-galactosidase expressed in cells with and without treatment in the same experiment.
86 4.2.4. RNR3-lacZ induction in post-replication repair deficient mutant strains
Post-replication repair is required by the cell to survive an S-phase with unrepaired lethal DNA lesions in the genome. After the replication machinery bypasses the damage, the PRR processes fill in the resulting gaps. InS. cerevisiae, members of the RAD6 pathway are responsible for PRR. Many lines of evidence suggested that the error-prone mechanism of PRR is not the preferred form of tolerance in eukaryotes (Broomfield et al., 2001 ). It is now clear that there are at least two independent error-free PRR pathways mediated by the product of RAD5 and POL30, and both of them are under the control of
RAD6/RAD18 (Xiao et al., 2000).
Rad6 is one of 13 ubiquitin-conjugating enzymes (Ubcs) in S. cerevisiae and is involved in diverse cellular functions. A rad6 null mutant is defective in PRR, resulting in an extreme sensitivity to a wide variety of DNA damaging agents and a defect in induced mutagenesis (Prakash et al., 1993). RAD6 is also necessary for sporulation
(Morrison et al., 1988), telomere silencing (Huang et al., 1997), and protein degradation based on the amino-end rule (Dohmen et al., 1991 ).
Like rad6 null mutants, rad18 null mutants are sensitive to UV, X-rays, y-rays, 4-
NQO, bleomycin and alkylating agents (Friedberg, 1991). However, it seems that
RAD18 is only involved in PRR. Physical interactions between Rad6 and Radl8 have been identified (Bailly et al., 1994). The Rad6-Rad18 heterodimer possesses a Ubc activity, a ssDNA binding activity and an ATPase activity (Bailly et al., 1997a). It has been postulated that the function of this heterodimer is similar to that of RecA in E. coli.
87 Radl8 binds ssDNA formed at a stalled replication fork and targets Rad6 to the site of damage (Bailly et al., 1997a; b)
Since PRR is centrally controlled by RAD6 and RAD18, in this study, UV radiation and the radiomimetic agent MMS were chosen to test RNR3-lacZ induction in rad6 and radl8 deletion mutant strains. Compared with wild type strains, the induction of RNR3- lacZ decreased dramatically after MMS treatment in both rad6 and radl8 null mutant strains (Fig. 4-1 0). Since the RNR3-lacZ induction in rad6 mutatants was compromised more severely than in radl8 cells, only rad18 cells were tested for its response to UV radiation in this study (Fig. 4-11 ).
88 60-r------~
T c 0 ..(,) :s "C c "C 0 LL
0 0.02 0.04
Methyl methanesulfonate {0/o)
Fig. 4-10. Comparison of the RNR3-lacZ induction in the wild type DBY747 (c) with that in the rad6 deletion mutant (•) and the rad18 deletion mutant (•) after MMS treatment. The yeast cells carrying pZZ2 (RNR3-lacZ) were incubated in SD-Ura medium, treated with MMS as indicated and incubated for additional four hours before assaying ~-galactosidase activity. The result was an average of three independent experiments. The fold of induction was calculated as a ratio of ~-galactosidase expressed in cells with and without treatment in the same experiment.
89 20-·~------~
c: 0 ..u ::s "C c: "C 0 LL
2 Ultraviolet light (J/m )
Fig. 4-11. Comparison of RNR3-lacZ induction in wild type DBY747 strains (•) with that in rad18 strain (c) after UV radiation treatment. The yeast cells carrying pZZ2 (RNR3-lacZ) were incubated in SD-Ura medium, treated with UV as indicated and incubated for additional four hours before assaying (3- galactosidase activity. The result was an average of three independent experiments. The fold of induction was calculated as a ratio of (3-galactosidase expressed in cells with and without treatment in the same experiment.
90 CHAPTER V. DISCUSSION
5.1. The yeast system is suitable for genotoxicity testing
Microorganism based short-term genotoxicity testing is important in the assessment of environmental toxins and carcinogens. The Ames test is used to determine whether an agent is a mutagen and the results from the Ames test correlate highly to its action as a rodent carcinogen (Ames et al., 1972; 1975; McCann et al., 1975; 1983).
Since its first development, it has been extensively employed for several decades. In general, this method is sensitive and convenient to perform, but is less effective in detecting DNA damaging agents that are mainly toxic but not mutagenic. A second method is based on the potential cytotoxicity of an agent. By performing a quantitative killing experiment, the toxicity of the test agent can be determined. The SOS Chromotest is based on cellular responses to DNA damage, especially based on the noticeable induction of gene expression. This method appears to be highly sensitive; however, a major concern is that the E. coli SOS system only responds to a specific type of DNA damage that results in single-stranded DNA recognized by RecA. Most importantly, both the Ames test and the SOS Chromotest employed prokaryotic cells. The differences between enkaryotes and prokaryotes and the fact that the well-known potent carcinogen
SDMH cannot be detected in bacterial systems have been discussed in Chapter I.
However, in the RNR3-lacZ system, SDMH showed high genotoxic potency and induced the RNR3 expression by more than 8-fold. This perhaps indicates that at least in some cases, yeast as a lower eukaryote is a more faithful model than bacteria to represent mammalian cells in mutagenic and geotoxicity tests. This is likely due to similar DNA
91 repair, tolerance and response pathways within eukaryotic cells which are different from
prokaryotic cells (Friedberg et al., 1995).
In this study, we examined the feasibility of employing the budding yeast DNA damage response to develop a microbial genotoxic testing system. The results allow us to
draw several conclusions. Firstly, our RNR3-lacZ testing system responded to treatment
with a broad range of DNA damaging agents and a DNA synthesis inhibitor, regardless of
whether they are mutagenic or genotoxic, whereas, all the tested non-genotoxic
compounds did not induce RNR3-lacZ expression. Secondly, the sensitivity of the RNR3-
lacZ system is comparable to that of SOS Chromotests. Thirdly, the reporter cassette can
be integrated into the host genome and achieve a stable inheritance, thus facilitating
applications beyond laboratory use. Fourthly, as a unicellular organism, S. cerevisiae
cells can be manipulated in a manner similar to bacterial cells. Finally, S. cerevisiae,
known as a baker's yeast, is biologically safe and environmentally friendly. In
combination with using lacZ as a reporter gene, it allows visual examination through a
color reaction and can be potentially developed into a field test.
5.2. RNR3 gene is the best choice among yeast DNA damage-inducible genes
Previous studies in our lab on the molecular mechanisms of cellular
transcriptional response to DNA-damaging agents (Xiao et al., 1993; Liu and Xiao, 1997;
Liu et al., 1997; Zhu and Xiao, 1998; 2001) led to this study to investigate the possibility
of using a yeast DNA damage-inducible gene as a reporter to detect genotoxic agents.
Results obtained from previous studies from our lab (Liu and Xiao, 1997) as well as other
92 laboratories (Elledge et al., 1993; Friedberg et al., 1995) indicate that, unlike the case in
E. coli, S. cerevisiae DNA damage-inducible genes appear to respond to different classes of DNA-damaging agents, making yeast more suitable for the development of a genotoxic testing system. Furthermore, it has been observed that different yeast genes not only have different levels of induction after DNA damage, but the kinetics may also be different (Zhu and Xiao, 1998). It was shown in this study that the RNR3 gene is probably the best choice among all yeast DNA damage-inducible genes studied to date
(see section 3-2-1). The RNR3 gene not only has one of the highest rates of induction after DNA damage, it is also induced at a much lower dose of DNA damage than most other genes. It is interesting to note that in a genome-wide investigation of DNA damage induction in budding yeast, some genes can be induced by MMS up to 250-fold (Jelinsky and Samson, 1999). However, under the same experimental conditions (0.1 o/o MMS for
60 minutes), RNR3 was only induced by up to 7-fold (Jelinsky and Samson, 1999). It was found that RNR3 induction reached a peak after 0.02% MMS treatment, and was severely inhibited by higher dose of MMS treatment. Nevertheless, the present results do not rule out the possibility that other yeast genes may be more sensitive to DNA damage
and more suitable for the genotoxic test than RNR3.
5.3. Quantitative assessment of the RNR3-lacZ testing system
5.3.1. Using of the "2-fold rule" to evaluate the results
In the Ames test, to determine whether or not the result is positive is a complex
question. This problem also exists in SOS chromotest and the present system. A number
93 of statistical methods and programs have been developed to analyze Ames test data
(Margolin et al., 1981; Bernstein et al., 1982; Simpson and Margolin, 1990; Edler, 1992).
An approach that has been widely used is referred to as "2-fold rule". The 2-fold method considers a compound significantly mutagenic if its mean number of revertants per plate at any dose is equal to or greater than twice the mean number of revertants per plate in the concurrent control (Cariello and Piegorsch, 1996). A non-statistical procedure has also been widely used to evaluate the results of Ames test (Zeiger et al., 1992). In this procedure, if a substance can produce a reproducible, dose-related increase than over the control group and the dose-related increase is less than two fold of the background, the chemical is considered as a weak mutagen. Most of the data evaluation methods are first used with Ames test. However, they are also applied to SOS system and other testing systems.
In this study, the 2-fold method was used to evaluate the results. There are at least two reasons for applying this method. Firstly, statistical methods tend to complicate the evaluation procedure and are obviously inappropriate for a system such as ours that is only designed for preliminary screening of genotoxic agents. Secondly, although it has been noticed that some aspects of the 2-fold method of analysis do not have a sound scientific foundation (Margolin, 1985; Cariello and Pieorsch, 1996), more than 40% of the recent publications and most of the publications from 1970's to 1990's on the Ames assay and SOS Chromotest still used this method for analysis (Kim and Margolin, 1999).
Hence, employing the same evaluation method makes the comparison with previous reports feasible.
94 In order to make the results more reliable, the variance of the experimental data in this study was maintained at a very low level (See Chapter III, Table 3-1 ).
5.3.2. Comparison of the RNR3-lacZ testing results with other genotoxicity testing
systems
Among the tested 14 agents, the two known non-genotoxic agents showed negative results in the RNR3-lacZ system. With regard to the other 12 agents, by the 2-fold rule, most of the results correlated with the Ames test and the SOS Chromotest very well
(Table 3-1). According to the results from MMS, EMS, HU and H20 2, the lowest response dose of this system is comparable to or even better than the SOS Chromotest
(See section 3-2-2). One agent (SDMH) showed positive result that was different from the standard Ames test which was discussed in Chapter III to demonstrate that it was one of the advantages of using the yeast system. The result from EtBr basically agreed with the SOS Chromotest and Ames test. It showed uncertain results in both Ames test
(Mamber et al., 1986) and SOS Chromotest (Mamber et al., 1986; Quillardet and
Hofnung, 1993), and the RNR3-lacZ system evaluated it as a non-genotoxic agent. ethidium bromide is an intercalating agent and commonly used as a non-radioactive marker to identify and visualize nucleic acid bands in electrophoresis and in other methods of gel-based nucleic acid separation. Intercalating agents often have a flat, multi-ring aromatic group of similar size as a nucleotide base pair. These compounds can insert or intercalate between adjacent base pairs, thus pushing the nucleotides apart.
When a single-stranded DNA containing an interacting molecule is used as template
95 during DNA replication, an extra nucleotide is often added to the newly synthesized chain, resulting in a + 1 frameshift mutation. If this process occurs during transcription, the DNA reading frame for RNA synthesis is changed and produces an altered protein.
However, it should be noted that not all the intercalating agents cause frameshift mutations. That is probably the reason why EtBr does not induce RNR3-lacZ expression.
Result obtained from RNR2-GFP and RAD54-GFP systems (Afanassiev et al., 2000) agree with the negative evaluation of EtBr. Another possible explanation for the negative results is the employment of the sensing genes. The corresponding sensing genes used in these systems may not respond to the specific DNA damage caused by EtBr. It remains possible that the use of appropriate sensing genes will lead to different results.
5.3.3. The threshold of genotoxicity tests
It has been commonly accepted that risk assessments of genotoxic chemicals are based on linear extrapolation methods and therefore the risk at low doses is considered to be directly proportional to that at high doses. However, there is substantial evidence that some chemicals may be genotoxic only at high doses by mechanisms that do not occur at low doses. In the present study, it was found that for some substances, the induction does not increase linearly (Fig. 3-2-1; Fig. 3-2-2). This may to some extent reflect the threshold dose for cells and the threshold dose may reflect the cellular repair capacity that undermines low dose DNA damage. In this study, a wide range of doses were tested to avoid the situation that all experimental doses were below the possible threshold dose. In general, the highest dose set for each agent was around LDso.
96 Several mechanisms may be responsible for the existence of threshold dose. In this study, the cell wall of yeast may reduce the availability of some types of chemicals inside cells and therefore delay the cellular response. Another possible mechanism is the organism's tolerance to DNA damage. During evolution, organisms, from prokaryotic organisms to both lower and higher eukaryotic organisms, developed different mechanisms to tolerate DNA damage at the cost of decreased genomic stability. In this system, the capability of tolerance may result in the non-responsiveness of cells to low dose treatment.
5.4. Improvement of the RNR3-lacZ system
5.4.1. Alteration of host strains to enhance the sensitivity of RNR3-lacZ system
In order to further enhance the sensitivity of the RNR3-lacZ system, several deletion
mutants defective in different DNA repair pathways were employed as host strains and
compared with wild type cells. All the mutant strains tested were created by a one-step
targeted gene deletion of the same parental strain DBY747 (Chen and Samson, 1991;
Xiao et al., 1996; 2001 ). Thus, the difference in the response to treatment can be solely
attributed to the gene( s) under investigation. The results indicated that the deletion of
DNA repair genes variably affected RNR3-lacZ induction by genotoxic agents.
Nevertheless, in some cases, the deletion of a DNA repair gene would enhance the
response of the RNR3-lacZ system to agents that primarily repaired by the corresponding
pathway, and this enhancement of sensitivity is potentially applicable to the improvement
of RNR3-lacZ testing system.
97 Comparing with wild type cells, the mag1 null mutation enhanced the sensitivity of the RNR3-lacZ system to MMS and EMS. Since MAGI encodes the DNA glycosylase that is specifically involved in the repair of alkylated lesions (Chen et al., 1989) and plays a critic role in the BER pathway, it is not surprising to observe increased RNR3-lacZ induction after treatment with alkylating agents. It indicates that inefficient repair of alkylated damage amplifies the messages sent to the regulation system and enhances the transcription of RNR3. In contrast, RNR3-lacZ induction in the magi mutant strain remains unaltered after UV treatment and dramatically decreased after y-ray treatment.
These results collectively suggest that the magi mutant strain can be used in the RNR3- lacZ test to detect DNA alkylating agents.
The Rad2 protein is critical for NER (Habraken et al., 1993). The mutation of its
human homologue, XP-G protein, results in hereditary caner. In this study, the RNR3-
lacZ induction dramatically increased in the rad2 null mutant strain after UV treatment.
The enhanced induction was also seen after treatment with a UV mimetic agent 4-NQO.
Eukaryotic NER is primarily responsible for the repair of DNA damage caused by UV,
bulky DNA adducts and DNA cross-links (Friedberg et al., 1995). The effects of the
rad2 mutation on RNR3-lacZ expression appear to agree with the hypothesis that
inactivation ofNER is able to enhance the detection of a broad range of genotoxic agents
that cause distortion of double-helix DNA structure. Our observations also agree with the
enhancement of Ames test by inactivaion of uvrB (Moreau et al., 1976). With regard to
MMS treatment, although maximum RNR3-lacZ induction in the rad2 mutant is lower
than in wild type cells, deletion of RAD2 does enhance RNR3-lacZ induction by MMS at
98 low doses. This result probably reflects the complicated involvement ofNER in the repair of DNA methylation damage in yeast cells (Xiao et al., 1996; 1998). Further investigation is needed for other DNA alkylating agents to assess the usefulness ofNER inactivation in the detection of this group of agents by the RNR3-lacZ system.
In this study, no mutant strain was found to enhance RNR3-lacZ induction after treatment with ionizing irradiation. In both of the rad50 and rad52 null mutant strains,
RNR3-lacZ induction decreased dramatically after y-ray treatment. It should be noted that although ionizing radiation has been extensively used to induce DSBs, DSBs are only a minor component of the radiation damage. The complex mixture of lesions makes it difficult to determine if a specific biological effect is caused by a given lesion.
It was found that after the deletion of certain DNA repair genes, the basal-level
RNR3-lacZ expression was increased. For example, in rad52 cells, the basal-level increased up to 7 fold after y-ray treatment. The enhanced basal level probably indicates the higher level of spontaneous DNA damage that leads to the de-repression of RNR3 gene expression. Indeed, RNR3-lacZ has been employed as a reporter of endogenous
DNA damage in yeast cells (Hryciw et al., 2002). For a genotoxicity testing system, not only is a high-level of gene expression (in this study referred to the specific J3-gal activity) desired, but the relative induction potential is also important.
5.4.2. Considerations to develop a more user-friendly RNR3-lacZ system
While our research was in progress, a testing system based on a GFP fusion to yeast DNA damage inducible promoters was reported (Afanassiev et al., 2000). The
99 authors found that the RNR2 fusion is more sensitive than the RAD54 fusion to DNA damage treatment. However, as hinted in previous studies (Elledge and Davis, 1989;
1990) and directly demonstrated in this study, the fold induction of RNR2 is much less than that of RNR3. Furthermore, the GFP fusion protocol described in that study requires overnight (14-16 hours) incubation of cells with the test agents even though RNR2-GFP was carried in a high-copy plasmid. Nevertheless, the idea of using a 96-well plate and automation is attractive, and may be complementary to our approach of employing a more sensitive RNR3-lacZ reporter and stable gene integration.
In fact, the SOS-Chromotest kit (EBPI, Ontario, Canada) has been in use since
1982. It is licensed from Institute Pasteur in France. The reporter used in this kit is the same as that of SOS-Chromotest, however, the reaction is carried out on a 96-well plate.
In addition to the reaction reagent, only a plate reader is needed. This is much faster and less costly than the RNR2-GFP system.
The present RNR3-lacZ test is carried out in test tubes. The advantage of this system is that it is time and cost saving. To make the system more user-friendly and suitable for field tests, during the present study, solid ~-gal assay was considered initially to replace the liquid assay. It was found that the replacement made the system even more complicated in treating the cells and quantifying the response. The SOS Chromotest kit inspired us to consider the possibility of developing the RNR3-lacZ system for field application.
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