DOCSLIB.ORG

Chapter 3. the Beginnings of Genomic Biology – Molecular

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Chapter 3. the Beginnings of Genomic Biology – Molecular Chapter 3. The Beginnings of Genomic Biology – Molecular Genetics Contents 3. The beginnings of Genomic Biology – molecular genetics 3.1. DNA is the Genetic Material 3.6.5. Translation initiation, elongation, and termnation 3.2. Watson & Crick – The structure of DNA 3.6.6. Protein Sorting in Eukaryotes 3.3. Chromosome structure 3.3.1. Prokaryotic chromosome structure 3.3.2. Eukaryotic chromosome structure 3.3.3. Heterochromatin & Euchromatin 3.4. DNA Replication 3.4.1. DNA replication is semiconservative 3.4.2. DNA polymerases 3.4.3. Initiation of replication 3.4.4. DNA replication is semidiscontinuous 3.4.5. DNA replication in Eukaryotes. 3.4.6. Replicating ends of chromosomes 3.5. Transcription 3.5.1. Cellular RNAs are transcribed from DNA 3.5.2. RNA polymerases catalyze transcription 3.5.3. Transcription in Prokaryotes 3.5.4. Transcription in Prokaryotes - Polycistronic mRNAs are produced from operons 3.5.5. Beyond Operons – Modification of expression in Prokaryotes 3.5.6. Transcriptions in Eukaryotes 3.5.7. Processing primary transcripts into mature mRNA 3.6. Translation 3.6.1. The Nature of Proteins 3.6.2. The Genetic Code 3.6.3. tRNA – The decoding molecule 3.6.4. Peptides are synthesized on Ribosomes CONCEPTS OF GENOMIC BIOLOGY Page 1 CHAPTER 3. THE BEGINNINGS OF GENOMIC BIOLOGY – MOLECULAR GENETICS (RETURN) 3.1. DNA IS THE GENETIC MATERIAL. (RETURN) As the development of classical genetics proceeded from Mendel in 1866 through the In 1928, a British scientist, early part of the 20th century the understanding Frederick Griffith, published his that Mendel’s factors that produced traits were work showing that live, rough, avirulent bacteria could be carried on chromosomes, and that there were transformed by a “principle” infinite ways that the genetic information from 2 found in dead, smooth, virulent parents could assort in each generation to bacteria into smooth, virulent produce the genetic variety demanded by bacteria. This meant that the Darwin’s theories on “origin of species” on which bacterial traits of rough versus natural selection acted. This gave rise to the Frederick Griffith smooth and avirulence versus study of gene behavior of more complex traits (1879-1941) viru- and an understanding of genes in populations. virulence were controlled by a substance that could carry the phenotype from dead to live cells. At the same time a quest for the material Griffith’s observations on Pneu-mococcus were inside a cell, perhaps a subcomponent of a controversial to say the least, and inspired a spirited chromosome, that carried the genetic instructions debate and much experimentation directed at proving to make organisms what they are was ongoing. whether the “transforming principle” was protein or nucleic acid, the two main components of CONCEPTS OF GENOMIC BIOLOGY Page 2 chromosomes identified early in the 20th century, well disrupted with a kitchen blender, and the location of before Griffith’s experiments. This debate continued the label determined. The 35S-labeled protein was found until Oswald Avery and his colleagues, Colin MacLeod, outside the infected cells, while the 32P-labeled DNA and Maclyn McCarty published their work in 1944 was inside the E. coli, indicating that DNA carried the unequivocally showing that DNA was, in fact, Griffith’s information needed for viral infection. transforming principle. This completely revolutionized genetics and is considered the founding observation of molecular genetics. Figure 3.1. An electron micrograph of bacteriophage T2 (left), and a sketch showing the structures present in the virus (right). The head consists of a DNA molecule surrounded by proteins, while the core, sheath, and tail fibers are all made of Oswald T. Avery Colin MacLeod Maclyn McCarty protein. Only the DNA molecule enters the cell. In 1953, more evidence supporting DNA being the genetic material resulted from the work of Alfred Once it was established that DNA was the genetic Hershey and Martha Chase on E. coli infected with material carrying the instructions for life so to speak, bacteriophage T2. In their experiment, T2 proteins attention turned to the question of “How could a were labeled with the 35S radioisotope, and T2 DNA was molecule carry genetic information?” The key to that labeled with was labeled with the 32P radioisotope. Then became obvious with a detailed understanding of the the labeled viruses were mixed separately with the E. structure of the DNA molecule, which was developed by coli host, and after a short time, phage attachment was two scientists a Cambridge University, James Watson and Francis Crick. CONCEPTS OF GENOMIC BIOLOGY Page 3 Rosalind Franklin a young x-ray crystallographer working in the laboratory of Maurice Wilkins at 3.2. WASON & CRICK – THE STRUCTURE OF DNA. Cambridge University used a technique known as x-ray (RETURN) diffraction to generate images of DNA molecules that showed that DNA had a helical structure with repeating The basic laboratory observations that lead to the structural elements every 0.34 nm and every 3.4 nm formulation of a structure for DNA did not involve along the axis of the molecule. biologists. Rather Irwin Chargaff, an analytical, organic chemist, and physicists, Rosalind Franklin and Maurice Wilkins made the laboratory observations that led to the solution of the structure of DNA. Chargaff determined that there were 4 different nitrogen bases found in DNA molecules; the purines, adenine (A) and guanine G), and the pyrimidines, cytosine (C) and thymine (T), and he purified DNA from a number of different sources so he could examine the quantitative relationships of A, T, G, and C. He con- Rosalind Franklin Maurice Wilkins cluded that in all DNA molecules, the mole-percentage of A was nearly equal to the mole-percentage of T, while the mole-percentage of G was nearly equal to the mole-percentage of C. Alternatively, you could state this as the mole-percentage of pyri-midine bases equaled the mole-percentage of purine bases. These observations became known as Chargaff’s rules. Figure 3.2. X-ray diffraction image of DNA molecule showing helical structure with repeat structural elements. CONCEPTS OF GENOMIC BIOLOGY Page 4 These astute observations allowed Watson and Crick The key elements of this structure are: to synthesize together a 3-dimentional structure of a • Double helical structure – each helix is made from DNA molecule with all of these essential features. This the alternating deoxyribose sugar and phosphate structure was published in 1953, and immediately groups derived from deoxynuclotides, which are the generated much excitement, culminating in a Nobel monomeric units that are used to make up Prize in Physiology and Medicine, in 1962 awarded to polymeric nucleic acid molecules. Each nucleotide Franklin, Wilkins, Watson, and Crick. in each chain consists of a nitrogen base of either the purine type (adenine or guanine) or the pyrimidine type (cytosine or thymidine) attached to the 1’-position of 2’-deoxyribose sugar, and a phosphate group, esterified by a phospho-ester bond to the 5’-position of the sugar. Figure 3.3. Watson & Crick’s DNA structure. Their model consisted of a double helicical structure with the sugars and phosphates making the two hlices on the outside of the structure. The sugars were held together by 3’-5’-phosphodiester bonds. The bases pair on the inside of the molecule with A always pairing with T, and G always pairing with C. This pairing leads to Chargaff’s observations about bases in DNA. Figure 3.4. Structures of purine and pyrimidine bases in DNA, and structure of 2’-deoxyribose sugar. CONCEPTS OF GENOMIC BIOLOGY Page 5 • The nucleotides are held together in sequence order Figure 3.5. The building bocks of nucleic along the length of the polynucleotide chain by 3’-5’- acids are nucleotides and nucleosides. Any phosphodiester bonds, and the strands demonstrate base together with a deoxyribose sugar forms a deoxyribonucleoside, while if the a polarity as the 5’-OH at one end of a polynuc- sugar is ribose a ribonucleoside is formed leotide strand is distinct from the 3’-OH at the other (not shown). Addition of a phosphate on end of the strand. Often, but not always, the 5’- the 5’ position of the sugar froms nucleotides from nucleosides. strand end will have a phosphate group attached. • Each of the 2 polynucleotide chains of the double helix are held together by hydrogen bonds beween the adenosines in one strand and the thymidines in the other strand, and between the guanosines in one strand hydrogen bonded to the cytosines in the other strand. Figure 3.6. Base pairing between A and T involves two hydrogen bonds, and pairing between G and C involves 3 Figure 3.7. Strand of DNA hydrogen bonds. This means that the forces holding strands showing the 3’,5’-phosphodi- together in G=C base pair-rich regions are stronger than in A=T ester bonds holding base pair-rich regions. nucleotides together. CONCEPTS OF GENOMIC BIOLOGY Page 6 In order to get a uniform diameter for the molecule and have proper alignment of the nucleotide pairs in the middle of the strands, the strands must be orient- ed in antiparallel fashion, i.e. with the strand polarity of each strand of the double helix going in the opposite direction (one strand is 3’-> 5’ whie the other is 5’ -> 3’). The truly elegant aspect of this solution to DNA structure produces a spacing of exactly 0.34 nm between nucleotide base pairs in the molecule, and there are 10 base-pairs per complete turn of the helix. This corresponds precisely with Rosalind Franklin’s x-ray diffraction measurements of repeating units of 0.34 nm and 3.4 nm, and with her measurements of 2 nm for the diameter of the double helix.
Load more

Recommended publications
  • Initiation of Enzymatic Replication at the Origin of the Escherichia
    Proc. Nati. Acad. Sci. USA Vol. 82, pp. 3954-3958, June 1985 Biochemistry Initiation of enzymatic replication at the origin of the Escherichia coli chromosome: Primase as the sole priming enzyme (DNA/orC/plasmids) ARIE VAN DER ENDEt, TANIA A. BAKER, TOHRU OGAWA*, AND ARTHUR KORNBERG Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305 Contributed by Arthur Kornberg, January 28, 1985 ABSTRACT The enzymatic replication of plasmids con- MATERIALS AND METHODS taining the unique (245 base pair) origin of the Escherichia coli chromosome (oriC) can be initiated with any of three enzyme DNAs and Reagents. pCM959 (4) was a gift from M. Meijer priming systems: primase alone, RNA polymerase alone, or (University of Amsterdam, The Netherlands); pTOA7 (T. both combined (Ogawa, T., Baker, T. A., van der Ende, A. & Ogawa) was constructed by inserting the Hae II-Acc I Kornberg, A. (1985) Proc. Natl. Acad. Sci. USA 82, oriC-containing fragment from M13oriC26 (7) via EcoRI 3562-3566). At certain levels of auxiliary proteins linkers into EcoRI-cleaved pMAPCdSG10, a deletion deriva- (topoisomerase I, protein HU, and RNase H), the solo primase tive of pBR327 (W. A. Segraves, personal communication); system is efficient and responsible for priming synthesis of all pSY317, M13oriC26, M13oriC2LB5, and M13AE101 are DNA strands. Replication of oriC plasmids is here separated described in Table 1 and elsewhere (3, 7). Tricine, creatine into four stages: (i) formation of an isolable, prepriming phosphate, ribo- and deoxyribonucleoside triphosphates complex requiring oriC, dnaA protein, dnaB protein, dnaC (rNTPs and dNTPs) were from Sigma; a-32P-labeled dTTP, protein, gyrase, single-strand binding protein, and ATP; (ii) rATP, rUTP, rGTP, and rCTP (>400 Ci/mmol; 1 Ci = 37 formation of a primed template by primase; (iii) rapid, GBq) were from Amersham.
    [Show full text]
  • Genome-Wide Detection of Chromosomal Rearrangements, Indels, and Mutations in Circular Chromosomes by Short Read Sequencing
    Downloaded from genome.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press Method Genome-wide detection of chromosomal rearrangements, indels, and mutations in circular chromosomes by short read sequencing Ole Skovgaard,1,3 Mads Bak,2 Anders Løbner-Olesen,1 and Niels Tommerup2 1Department of Science, Systems and Models, Roskilde University, DK-4000 Roskilde, Denmark; 2Wilhelm Johannsen Centre for Functional Genome Research, Department of Cellular and Molecular Medicine, University of Copenhagen, DK-2200 Copenhagen, Denmark Whole-genome sequencing (WGS) with new short-read sequencing technologies has recently been applied for genome- wide identification of mutations. Genomic rearrangements have, however, often remained undetected by WGS, and additional analyses are required for their detection. Here, we have applied a combination of WGS and genome copy number analysis, for the identification of mutations that suppress the growth deficiency imposed by excessive initiations from the Escherichia coli origin of replication, oriC. The E. coli chromosome, like the majority of bacterial chromosomes, is circular, and DNA replication is initiated by assembling two replication complexes at the origin, oriC. These complexes then replicate the chromosome bidirectionally toward the terminus, ter. In a population of growing cells, this results in a copy number gradient, so that origin-proximal sequences are more frequent than origin-distal sequences. Major rearrangements in the chromosome are, therefore, readily identified by changes in copy number, i.e., certain sequences become over- or under-represented. Of the eight mutations analyzed in detail here, six were found to affect a single gene only, one was a large chromosomal inversion, and one was a large chromosomal duplication.
    [Show full text]
  • Arthur Kornberg Discovered (The First) DNA Polymerase Four
    Arthur Kornberg discovered (the first) DNA polymerase Using an “in vitro” system for DNA polymerase activity: 1. Grow E. coli 2. Break open cells 3. Prepare soluble extract 4. Fractionate extract to resolve different proteins from each other; repeat; repeat 5. Search for DNA polymerase activity using an biochemical assay: incorporate radioactive building blocks into DNA chains Four requirements of DNA-templated (DNA-dependent) DNA polymerases • single-stranded template • deoxyribonucleotides with 5’ triphosphate (dNTPs) • magnesium ions • annealed primer with 3’ OH Synthesis ONLY occurs in the 5’-3’ direction Fig 4-1 E. coli DNA polymerase I 5’-3’ polymerase activity Primer has a 3’-OH Incoming dNTP has a 5’ triphosphate Pyrophosphate (PP) is lost when dNMP adds to the chain E. coli DNA polymerase I: 3 separable enzyme activities in 3 protein domains 5’-3’ polymerase + 3’-5’ exonuclease = Klenow fragment N C 5’-3’ exonuclease Fig 4-3 E. coli DNA polymerase I 3’-5’ exonuclease Opposite polarity compared to polymerase: polymerase activity must stop to allow 3’-5’ exonuclease activity No dNTP can be re-made in reversed 3’-5’ direction: dNMP released by hydrolysis of phosphodiester backboneFig 4-4 Proof-reading (editing) of misincorporated 3’ dNMP by the 3’-5’ exonuclease Fidelity is accuracy of template-cognate dNTP selection. It depends on the polymerase active site structure and the balance of competing polymerase and exonuclease activities. A mismatch disfavors extension and favors the exonuclease.Fig 4-5 Superimposed structure of the Klenow fragment of DNA pol I with two different DNAs “Fingers” “Thumb” “Palm” red/orange helix: 3’ in red is elongating blue/cyan helix: 3’ in blue is getting edited Fig 4-6 E.
    [Show full text]
  • Molecular Biology and Applied Genetics
    MOLECULAR BIOLOGY AND APPLIED GENETICS FOR Medical Laboratory Technology Students Upgraded Lecture Note Series Mohammed Awole Adem Jimma University MOLECULAR BIOLOGY AND APPLIED GENETICS For Medical Laboratory Technician Students Lecture Note Series Mohammed Awole Adem Upgraded - 2006 In collaboration with The Carter Center (EPHTI) and The Federal Democratic Republic of Ethiopia Ministry of Education and Ministry of Health Jimma University PREFACE The problem faced today in the learning and teaching of Applied Genetics and Molecular Biology for laboratory technologists in universities, colleges andhealth institutions primarily from the unavailability of textbooks that focus on the needs of Ethiopian students. This lecture note has been prepared with the primary aim of alleviating the problems encountered in the teaching of Medical Applied Genetics and Molecular Biology course and in minimizing discrepancies prevailing among the different teaching and training health institutions. It can also be used in teaching any introductory course on medical Applied Genetics and Molecular Biology and as a reference material. This lecture note is specifically designed for medical laboratory technologists, and includes only those areas of molecular cell biology and Applied Genetics relevant to degree-level understanding of modern laboratory technology. Since genetics is prerequisite course to molecular biology, the lecture note starts with Genetics i followed by Molecular Biology. It provides students with molecular background to enable them to understand and critically analyze recent advances in laboratory sciences. Finally, it contains a glossary, which summarizes important terminologies used in the text. Each chapter begins by specific learning objectives and at the end of each chapter review questions are also included.
    [Show full text]
  • Dna Replication in Archaea: Priming, Transferase, and Elongation Activities
    DNA REPLICATION IN ARCHAEA: PRIMING, TRANSFERASE, AND ELONGATION ACTIVITIES by Zhongfeng Zuo Bachelor degree, Beijing Technology and Business University, 1999 Submitted to the Graduate Faculty of the Kenneth P. Dietrich School of Arts and Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2012 UNIVERSITY OF PITTSBURGH THE KENNETH P. DIETRICH SCHOOL OF ARTS AND SCIENCES This dissertation was presented by Zhongfeng Zuo It was defended on January 27, 2012 and approved by Stephen G. Weber, Professor, Department of Chemistry Billy Day, Professor, Department of Chemistry, Department of Pharmacy Renã A. S. Robinson, Assistant Professor, Department of Chemistry Dissertation Advisor: Michael A. Trakselis, Assistant Professor, Department of Chemistry ii DNA REPLICATION IN ARCHAEA: PRIMING, TRANSFERASE, AND ELONGATION ACTIVITIES Zhongfeng Zuo, Ph.D University of Pittsburgh, 2012 Copyright © by Zhongfeng Zuo 2012 iii DNA REPLICATION IN ARCHAEA: PRIMING, TRANSFERASE, AND ELONGATION ACTIVITIES Zhongfeng Zuo, Ph.D University of Pittsburgh, 2012 We have biochemically characterized the bacterial-like DnaG primase contained within the hyperthermophilic crenarchaeon Sulfolobus solfataricus (Sso ) and compared in vitro priming kinetics with those of the eukaryotic-like primase (PriS&L) also found in Sso . Sso DnaG exhibited metal- and temperature-dependent profiles consistent with priming at high temperatures. The distribution of primer products for Sso DnaG was discrete but highly similar to the distribution of primer products produced by the homologous Escherichia coli DnaG. The predominant primer length was 13 bases, although less abundant products of varying sizes are also present. Sso DnaG was found to bind DNA cooperatively as a dimer with a moderate dissociation constant.
    [Show full text]
  • Micropropagation, Genetic Engineering, and Molecular Biology Expression Vectors Containing the Gene(S) and Promoter(S) of Populus
    This file was created by scanning the printed publication. Errors identified by the software have been corrected; however, some errors may remain. Chapter 11 Growth and Development Alteration in Transgenic Populus: Status and Potential Applications1 Bjorn Sundberg, Hannele Tuominen, Ove Nilsson, Thomas Moritz, c. H. Anthony Little, Goran Sandberg, and Olof Olsson Introduction Using Transgenic Populus to Study Growth and Development With the development of gene-transfer techniques appli­ cable to forest tree species, genetic engineering is becoming in Woody Species an alternative to traditional tree breeding. To date, routine transformation methods for several hardwood species, par­ The best model system for understanding the genetic ticularly Populus and Eucalyptus, have been established and and physiological control of tree growth and wood for­ promising advances have occurred in the development ~f mation is a perennial species containing a vascular cam­ transformation protocols for conifers (Olarest et al. 1996; Ellis bium, which is the meristem that produces secondary et al. 1996; Jouanin et al. 1993; Walters et al. 1995). Rapid xylem and phloem. Presently, Populus is the preferred tree­ progress in transformation technology makes it possible to model system because it has several useful fea.tures. develop genetic engineering tools that modify economically Populus has a small genome, approximately 5 x lOS base tractable parameters related to growth and yield in tree spe­ pairs (bp ), which encourages molecular mapping, library cies. Such work will also increase our understanding of ge­ screening, and rescue cloning. Saturated genetic maps are netic and physiological regulation of growth and already constructed for several Populus spp. (Bradshaw et development in woody species.
    [Show full text]
  • Recruitment of Terminal Protein to the Ends of Streptomyces Linear Plasmids and Chromosomes by a Novel Telomere-Binding Protein Essential for Linear DNA Replication
    Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Recruitment of terminal protein to the ends of Streptomyces linear plasmids and chromosomes by a novel telomere-binding protein essential for linear DNA replication Kai Bao1 and Stanley N. Cohen1,2,3 1Department of Genetics and 2Department of Medicine, Stanford University School of Medicine, Stanford, California 94305-5120, USA Bidirectional replication of Streptomyces linear plasmids and chromosomes from a central origin produces unpaired 3؅-leading-strand overhangs at the telomeres of replication intermediates. Filling in of these overhangs leaves a terminal protein attached covalently to the 5؅ DNA ends of mature replicons. We report here the essential role of a novel 80-kD DNA-binding protein (telomere-associated protein,Tap) in this process. Biochemical studies,yeast two-hybrid analysis,and immunopre cipitation/immunodepletion ,experiments indicate that Tap binds tightly to specific sequences in 3؅ overhangs and also interacts with Tpg bringing Tpg to telomere termini. Using DNA microarrays to analyze the chromosomes of tap mutant bacteria,we demonstrate that survivors of Tap ablation undergo telomere deletion,chromosome circularization,and amplification of subtelomeric DNA. Microarray-ba sed chromosome mapping at single-ORF resolution revealed common endpoints for independent deletions,identi fied amplified chromosomal ORFs adjacent to these endpoints,and quantified the copy number of these ORFs. Sequence analysis confirmed chromosome circularization and revealed the insertion of adventitious DNA between joined chromosome ends. Our results show that Tap is required for linear DNA replication in Streptomyces and suggest that it functions to recruit and position Tpg at the telomeres of replication intermediates.
    [Show full text]
  • Glossary/Index
    Glossary 03/08/2004 9:58 AM Page 119 GLOSSARY/INDEX The numbers after each term represent the chapter in which it first appears. additive 2 allele 2 When an allele’s contribution to the variation in a One of two or more alternative forms of a gene; a single phenotype is separately measurable; the independent allele for each gene is inherited separately from each effects of alleles “add up.” Antonym of nonadditive. parent. ADHD/ADD 6 Alzheimer’s disease 5 Attention Deficit Hyperactivity Disorder/Attention A medical disorder causing the loss of memory, rea- Deficit Disorder. Neurobehavioral disorders character- soning, and language abilities. Protein residues called ized by an attention span or ability to concentrate that is plaques and tangles build up and interfere with brain less than expected for a person's age. With ADHD, there function. This disorder usually first appears in persons also is age-inappropriate hyperactivity, impulsive over age sixty-five. Compare to early-onset Alzheimer’s. behavior or lack of inhibition. There are several types of ADHD: a predominantly inattentive subtype, a predomi- amino acids 2 nantly hyperactive-impulsive subtype, and a combined Molecules that are combined to form proteins. subtype. The condition can be cognitive alone or both The sequence of amino acids in a protein, and hence pro- cognitive and behavioral. tein function, is determined by the genetic code. adoption study 4 amnesia 5 A type of research focused on families that include one Loss of memory, temporary or permanent, that can result or more children raised by persons other than their from brain injury, illness, or trauma.
    [Show full text]
  • Chromosome Duplication in Saccharomyces Cerevisiae
    | YEASTBOOK GENOME ORGANIZATION AND INTEGRITY Chromosome Duplication in Saccharomyces cerevisiae Stephen P. Bell*,1 and Karim Labib†,1 *Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, and yMedical Research Council Protein Phosphorylation and Ubiquitylation Unit, Sir James Black Centre, School of Life Sciences, University of Dundee, DD1 5EH, United Kingdom ORCID ID: 0000-0002-2876-610X (S.P.B.) ABSTRACT The accurate and complete replication of genomic DNA is essential for all life. In eukaryotic cells, the assembly of the multi-enzyme replisomes that perform replication is divided into stages that occur at distinct phases of the cell cycle. Replicative DNA helicases are loaded around origins of DNA replication exclusively during G1 phase. The loaded helicases are then activated during S phase and associate with the replicative DNA polymerases and other accessory proteins. The function of the resulting replisomes is monitored by checkpoint proteins that protect arrested replisomes and inhibit new initiation when replication is inhibited. The replisome also coordinates nucleosome disassembly, assembly, and the establishment of sister chromatid cohesion. Finally, when two replisomes converge they are disassembled. Studies in Saccharomyces cerevisiae have led the way in our understanding of these processes. Here, we review our increasingly molecular understanding of these events and their regulation. KEYWORDS DNA replication; cell cycle; chromatin; chromosome duplication; genome stability;
    [Show full text]
  • Changing Perspectives on the Role of Dnaa-ATP in Orisome Function and Timing Regulation
    fmicb-10-02009 August 28, 2019 Time: 17:19 # 1 REVIEW published: 29 August 2019 doi: 10.3389/fmicb.2019.02009 Changing Perspectives on the Role of DnaA-ATP in Orisome Function and Timing Regulation Alan C. Leonard1*, Prassanna Rao2, Rohit P. Kadam1 and Julia E. Grimwade1 1 Laboratory of Microbial Genetics, Department of Biomedical and Chemical Engineering and Science, Florida Institute of Technology, Melbourne, FL, United States, 2 Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, United States Bacteria, like all cells, must precisely duplicate their genomes before they divide. Regulation of this critical process focuses on forming a pre-replicative nucleoprotein complex, termed the orisome. Orisomes perform two essential mechanical tasks that configure the unique chromosomal replication origin, oriC to start a new round of chromosome replication: (1) unwinding origin DNA and (2) assisting with loading of the replicative DNA helicase on exposed single strands. In Escherichia coli, a necessary orisome component is the ATP-bound form of the bacterial initiator protein, DnaA. DnaA- ATP differs from DnaA-ADP in its ability to oligomerize into helical filaments, and in its ability to access a subset of low affinity recognition sites in the E. coli replication origin. Edited by: The helical filaments have been proposed to play a role in both of the key mechanical Ludmila Chistoserdova, tasks, but recent studies raise new questions about whether they are mandatory for University of Washington, allADP United States orisome activity. It was recently shown that a version of E. coli oriC (oriC ), whose Reviewed by: multiple low affinity DnaA recognition sites bind DnaA-ATP and DnaA-ADP similarly, was Anders Løbner-Olesen, fully occupied and unwound by DnaA-ADP in vitro, and in vivo suppressed the lethality University of Copenhagen, Denmark of DnaA mutants defective in ATP binding and ATP-specific oligomerization.
    [Show full text]
  • Change in Chromosome Number Associated with a Double Deletion in the Neurospora Crussa Mitochondrial Chromosome
    Copyright 0 1989 by the Genetics Society of America Change in Chromosome Number Associated With a Double Deletion in the Neurospora crussa Mitochondrial Chromosome Samson R. Gross, Ann Mary and Pearl H. Levine Department of Biochemistry, Division of Genetics, Duke University, Durham, North Carolina 27710 Manuscript received October 27, 1988 Accepted for publication December 19, 1988 ABSTRACT The mitochondrial genome of Neurospora is usually found in a single covalently closed circular 62-kbp DNA molecule. We report here that the mitochondrial genome of a phenotypic revertant of a stopper mutant (stp-ruv) is contained primarily in two separate, nonoverlapping, autonomously replicating circular chromosomes. The circles, one about 21 kbp and the other somewhat less than 36 kbp are derived from the most frequent classes of recombinant chromosomes (21 and 41 kbp) in the chromosomal population of mitochondria in the original stopper mutant. The new, more stable chromosomal configuration, is associated with the deletion of two sequences (1 kbp and 4 kbp) at the splice junctions of the two circles. The data suggest that both deletions are likely to have originated from a single recombinational event involved in generating the 36-kbp circle. Secondary, sponta- neously arising derivatives of stp-ruv have been found to yield, at high copy number, shortsections of the 21-kbp circle in covalently closed supercoiled circles varying from unit length to very high multimers. The amplified segments span a common segment likely to contain the replication origin of the 2 1-kbp chromosome. - NTRACHROMOSOMAL recombination is a fre- and LEVINE1984). As indicated in the following sec- I quent event during the normal growth and repli- tions, the stability of the n = 2 chromosomal comple- cation of mitochondria in many different plant and ment is associated with the loss of two extended se- fungal species (CUMMINGS,BELCOUR and GRAND- quences of the single chromosome of normal mito- CHAMP 1979; PALMERand SHIELDS1984; GROSS, chondria.
    [Show full text]
  • Cryptic Single-Stranded-DNA Binding Activities of the Phage P And
    Proc. Natl. Acad. Sci. USA Vol. 94, pp. 1154–1159, February 1997 Biochemistry Cryptic single-stranded-DNA binding activities of the phage l P and Escherichia coli DnaC replication initiation proteins facilitate the transfer of E. coli DnaB helicase onto DNA (phage l DNA replicationyE. coli DNA replicationyregulation of DNA helicase action) BRIAN A. LEARN,SOO-JONG UM*, LI HUANG†, AND ROGER MCMACKEN‡ Department of Biochemistry, School of Hygiene and Public Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, MD 21205 Communicated by Thomas Kelly, Johns Hopkins University, Baltimore, MD, December 5, 1996 (received for review October 2, 1996) ABSTRACT The bacteriophage l P and Escherichia coli tight complex with the hexameric DnaB helicase (16) and the DnaC proteins are known to recruit the bacterial DnaB repli- helicase is recruited to the viral origin through interactions of the cative helicase to initiator complexes assembled at the phage and PzDnaB complex with the O-some (7, 11, 12). This second-stage bacterial origins, respectively. These specialized nucleoprotein nucleoprotein structure is seemingly unreactive until it is partially assemblies facilitate the transfer of one or more molecules of disassembled by the action of the E. coli DnaJ, DnaK, and GrpE DnaB helicase onto the chromosome; the transferred DnaB, in molecular chaperone system (8–10, 17). This disassembly reac- turn, promotes establishment of a processive replication fork tion stimulates DnaB helicase action by freeing DnaB from its apparatus. To learn more about the mechanism of the DnaB strong association with the P protein, an interaction which is transfer reaction, we investigated the interaction of replication known to suppress the ATPase and helicase activities of DnaB initiation proteins with single-stranded DNA (ssDNA).
    [Show full text]