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Variant requirements for DNA repair proteins in cancer cell lines that use alternative lengthening of telomere mechanisms of elongation

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By
Alaina Rae Martinez
Biomedical Sciences Graduate Program

The Ohio State University
2016

Dissertation Committee:
Dr. Jeffrey D. Parvin, Advisor
Dr. Joanna Groden Dr. Amanda E. Toland Dr. Kay F. Huebner
Copyright by
Alaina Rae Martinez
2016

Abstract

The human genome relies on DNA repair proteins and the telomere to maintain genome stability. Genome instability is recognized as a hallmark of cancer, as is limitless replicative capacity. Cancer cells require telomere maintenance to enable this uncontrolled growth. Most often telomerase is activated, although a subset of human cancers depend on recombination-based mechanisms known as Alternative Lengthening of Telomeres (ALT). ALT depends invariably on recombination and its associated DNA repair proteins to extend telomeres. This study tested the hypothesis that the requirement for those requisite recombination proteins include other types of DNA repair proteins. These functions were tested in ALT cell lines using C-circle abundance as a marker of ALT. The requirement for homologous recombination proteins and other DNA repair proteins varied between ALT cell lines compared. Several proteins essential for homologous recombination were dispensable for C-circle production in some ALT cell lines, while proteins grouped into excision DNA repair processes were required for C- circle production. The MSH2 mismatch repair protein was required for telomere recombination by intertelomeric exchange. In sum, our study suggests that ALT proceeds by multiple mechanisms that differ between human cancer cell lines and that some of these depend on DNA repair proteins not associated with homologous recombination pathways. Further studies of all DNA repair pathways in ALT will likely lead to a better understanding of ALT mechanisms and ultimately better ALT-targeted therapeutics. ii

Dedication

To my grandparents, parents and husband who have worked hard and made sacrifices so that I could have this opportunity and to my siblings for being examples for me to follow.

iii

Acknowledgments

I would like to acknowledge my advisors Dr. Jeffrey Parvin and Dr. Joanna
Groden for their guidance and encouragement throughout graduate school. I would like to thank committee members Dr. Amanda Toland and Dr. Kay Huebner for their helpful suggestions, interest in my project and thought-provoking questions. I would like to thank the 9th floor of the BRT; they have been a very collegial and friendly group of cancer researchers. Over the years there have been many members of the Parvin and Groden labs and rotation labs that I have been lucky to have met. Many became not only friends but

my “Columbus family” who supported me through the hard times. I am so grateful for

those friendships and could not have made it through graduate school without them.
I would also like to thank those who have guided me to this point in my life: teachers from Lewiston-Porter; professors from The College of Wooster, who believed in my capabilities; Dr. José Lemos who provided me the same opportunities, as a lab technician, as he did his students and prepared me for grad school; to him, Dr. Jacqueline Abranches and Dr. Jessica Kajfasz, who sparked my interest in pursuing a PhD; Lemos lab friends that lent empathetic ears and encouraged me. I thank my family and friends for support, love and belief in me. Lastly, I would like to thank my husband, Senyo. It was difficult to be away from each other for five years but he encouraged me to finish graduate school. I look forward to spending the rest of my life with him, finally!

iv

Vita

May 2007 .......................................................B.A. Biochemistry & Molecular Biology,
The College of Wooster

2010 to present ..............................................PhD Candidate, The Ohio State University

Publications

Acharya, S., Kaul, Z., Gocha, A.S., Martinez, A.R., Harris, J., Parvin, J.D., and Groden,
J. (2014). Association of BLM and BRCA1 during telomere maintenance in ALT cells. PLoS One 9: 1–13.
Singh, M., Martinez, A.R., Lee, B.S. (2013). HuR inhibits apoptosis by amplifying Akt signaling through a positive feedback loop. J Cell Physio 228: 182-189.
Abranches, J., Miller, J.H., Martinez, A.R., Simpson-Haidaris, P.J., Burne, R.A., Lemos,
J.A. (2011). The collagen-binding protein Cnm is required for Streptococcus mutans adherence to and intracellular invasion of human coronary artery endothelial cells. Infect and Immun 79: 2277-2284.
Martinez, A.R., Abranches, J., Kajfasz, J.K., Lemos, J.A. (2010). Characterization of the
S. sobrinus acid-stress response by interspecies microarrays and proteomics. Mol Oral Micro 25: 331-342.
Kajfasz, J.K., Rivera-Ramos, I., Abranches, J., Martinez, A.R., Rosalen, P.L., Derr,
A.M., Quivey R.G., Lemos, J.A. (2010). Global regulation by two Spx proteins modulate stress tolerance, survival, and virulence in S. mutans. J Bacteriol 192: 2546-2556.
Abranches, J., Martinez, A.R., Kajfasz, J.K., Chávez, V., Garsin, D.A., Lemos, J.A.
(2009). The molecular alarmone (p)ppGpp mediates stress responses, Vancomycin tolerance, and virulence in Enterococcus faecalis. J Bacteriol 191: 2248-2256.
Kajfasz, J.K., Martinez, A.R., Rivera-Ramos, I., Abranches, J., Koo, H., Quivey, Jr.,
R.G., Lemos, J.A. (2009). Role of Clp proteins in the expression of virulence properties of Streptococcus mutans. J Bacteriol 191: 2060-2068.

Fields of Study

Major Field: Biomedical Sciences v

Table of Contents

Abstract............................................................................................................................... ii Dedication..........................................................................................................................iii Acknowledgments.............................................................................................................. iv Vita...................................................................................................................................... v Publications......................................................................................................................... v List of Tables ..................................................................................................................... ix List of Figures..................................................................................................................... x Chapter 1: Functions of DNA Repair Proteins in Alternative Lengthening of Telomere Mechanisms ........................................................................................................................ 1

I. Telomere maintenance................................................................................................. 2
I.1 Telomere structure and function ............................................................................ 2 I.2 Telomerase ............................................................................................................. 4 I.3 Alternative lengthening of telomeres ..................................................................... 4
II. DNA repair proteins in ALT ...................................................................................... 9
II.1 DNA damage sensors.......................................................................................... 10 II.2 Chromatin modifiers ........................................................................................... 12 vi
II.3 Double-strand break proteins.............................................................................. 13 II.4 RecQ-like helicases............................................................................................. 18 II.5 Excision repair .................................................................................................... 19
Chapter 2: Thesis Rationale and Research Objectives ..................................................... 26 Chapter 3: Differential Requirements for DNA Repair Proteins in Cell Lines Using Alternative Lengthening of Telomere Mechanisms ......................................................... 29

I. Introduction................................................................................................................ 29 II. Materials and methods.............................................................................................. 32
Cell line s.................................................................................................................... 32 siRNA knockdow n...................................................................................................... 32 Western blots ............................................................................................................. 33 qRT PCR assays......................................................................................................... 33 C-circle assays........................................................................................................... 33 Telomere sister chromatid exchange assays ............................................................. 34 Homology-directed repair assays.............................................................................. 35
III. Results..................................................................................................................... 37

BRCA1 depletion in five ALT cell lines decreases C-circles, a quantifiable marker of

ALT ............................................................................................................................ 37 vii

ALT cells do not depend on all requisite homologous recombination proteins for telomere maintenance although requirements differ between ALT-positive cell lines

................................................................................................................................... 42

Non-homologous end joining proteins are not critical for ALT................................ 47 Nucleotide excision repair, DNA mismatch repair, and base excision repair proteins

can contribute to AL T................................................................................................ 47

MSH2 stimulates intertelomeric exchanges in U-2 OS ALT cells and MSH2 and MPG affect homology-directed repair in HeLa cells ................................................ 50

IV. Discussion............................................................................................................... 55
Chapter 4: Thesis Summary and Future Directions.......................................................... 59
I. Thesis summary ........................................................................................................ 59 II. Future directions....................................................................................................... 63 III. Significance of understanding mechanisms of ALT............................................... 75
References......................................................................................................................... 77

viii

List of Tables

Table 1. Sequence of siRNAs and quantitative PCR primers............................................ 36 Table 2. Summary of data from DNA repair depletions and C-circle measurements in ALT cells .......................................................................................................................... 58

ix

List of Figures

Figure 1: T-loop with shelterin complex. ........................................................................... 3 Figure 2: ALT cells have distinct and characteristic cytological phenotypes. ................... 7 Figure 3: C-circle assay (CC assay).................................................................................... 9 Figure 4: DNA damage sensing and recruitment of proteins to a double-strand break.... 11 Figure 5: Homologous recombination DNA repair pathway............................................ 15 Figure 6: Non-homologous end joining DNA repair pathway. ........................................ 17 Figure 7: Nucleotide excision DNA repair pathway......................................................... 21 Figure 8: Mismatch DNA repair pathway. ....................................................................... 23 Figure 9: Base excision DNA repair pathway. ................................................................. 24 Figure 10: Depletion of BRCA1 reduces C-circles in five ALT cell lines but not in a telomerase-dependent cell line.......................................................................................... 39 Figure 11: Knockdown of BRCA1 using a second siRNA reduces C-circles in ALT cell lines and confirms previous experiments.......................................................................... 41 Figure 12: Depletion of homologous recombination proteins BARD1, BRCA2, PALB2 or WRN in ALT cells do not alter C-circle abundance......................................................... 43 Figure 13: Depletion of RNF8 or RNF168 lowers C-circle levels in VA-13 cells and depletion of NHEJ proteins does not alter C-circle abundance in Saos-2, U-2 OS or VA- 13 cells. ............................................................................................................................. 46

x
Figure 14: Depletion of XPA, MSH2 or MPG lowers C-circle levels in U-2 OS and VA- 13 cells. ............................................................................................................................. 49 Figure 15: Depletion of MSH2, XPA or MPG lowers intertelomeric exchanges in U-2 OS cells............................................................................................................................. 52 Figure 16: Depletion of MSH2 (MMR) or MPG (BER) proteins decrease homologydirected repair (HDR). ...................................................................................................... 54 Figure 17: Variant requirements for DNA repair proteins in ALT................................... 62

xi

Chapter 1: Functions of DNA Repair Proteins in Alternative Lengthening of
Telomere Mechanisms

Maintenance of genome integrity is a critical process in human cells as select unrepaired mutations cause genome instability which can precipitate human diseases such as premature aging, chronic pulmonary disease and cancer. Human cells are faced with the challenge of repairing tens of thousands of DNA lesions per day in order to preserve genome integrity. DNA repair relies on concerted functions from a host of proteins: DNA damage sensors, DNA damage signaling and cell cycle checkpoint proteins, mediator proteins, and nucleases, polymerases, helicases and ligases (Jackson and Bartek, 2009). Some of these proteins function not only to protect DNA in the genome but also DNA found at the end of chromosomes that compose the telomere. The telomere is a DNA- protein structure that also functions to maintain genome integrity as it protects genomic DNA from deterioration and prevents DNA ends from eliciting a DNA damage response. DNA repair proteins and the telomere are linked in their ability to maintain genome integrity. In some cancer cell lines and tumor types, DNA repair proteins are required for mechanisms of Alternative Lengthening of Telomeres (ALT). Activation of this telomere lengthening maintenance mechanism(s) in cells permits uncontrolled proliferation, which is necessary for the onset of cancer.

1

I. Telomere maintenance
I.1 Telomere structure and function

Normal human cells have functions and structures that prevent the cell from becoming neoplastic. One key structure is the telomere, in humans, which is a noncoding stretch of DNA made up of 5′TTAGGG3′ and complementary 5′CCCTAA3′ repeats at the end of a chromosome. Telomeres can range from 10-14 kb in length in normal cells and have a 3′ G-rich over-hang of 130-210 base-pairs in length (de Lange et al., 1990; Makarov et al., 1997). The G-rich overhang is sufficient for preventing end-to-end chromosome fusions, but if left in a linear confirmation it would be detected as a doublestrand break (DSB) (Zhu et al., 2003). To prevent this, the telomere forms a “t-loop” in which the 3′ G-rich overhang invades the double strand portion of the telomere forming a D-loop (Griffith et al., 1999). A complex of proteins that binds the telomere, called the

shelterin complex, helps facilitate this t-loop and also helps “shelter” the telomere from

the DNA damage response. The shelterin complex consists of telomere binding proteins, the telomere repeat binding factors 1 (TRF1) and 2 (TRF2), and the single-stranded telomere binding protein, protection of telomeres 1 (POT1). The proteins TRF1- interacting nuclear factor 2 (TIN2), tripeptidyl peptidase 1 (TPP1) and Ras-proximate protein 1 (RAP1) facilitate the telomere-binding protein interactions in the complex (Figure 1) (de Lange, 2005). Proteins involved in DNA repair and DNA damage signaling also associate with the telomere but are unable to trigger the DNA damage response or cell cycle arrest (Matulić et al., 2007; Zhu et al., 2000).

2

Figure 1: T-loop with shelterin complex.

Shelterin proteins facilitate looping of the telomere DNA (t-loop). DNA and proteins to the right of the // represent the telomeric region.

The telomere is essential for protecting genomic DNA from deteriorating during the life-cycle of the cell. With each cell division, the DNA replication machinery cannot fully replicate the very end of the telomere; this is known as the “end replication problem”. There is a steady loss of 50-150 base-pairs of DNA per cell division. (Makarov et al., 1997; Sfeir and de Lange, 2012). Eventually, when a cell has undergone numerous

cell divisions, the telomere becomes “critically short”. Consequently, the telomere can no

longer perform its function; shelterin proteins can no longer bind; senescence is induced. If genetic or epigenetic changes occur such that cell-cycle checkpoint proteins are altered in a cell, the cell will continue to proliferate even with this extreme telomere dysfunction.
3
When the telomere reaches about 1.5 kilobases in length, “crisis” occurs, in which cytogenetic abnormalities or cell death ensue (Counter et al., 1992; Greenberg, 2005; Hemann et al., 2001; Kim et al., 1994). The chromosome instability that occurs at “crisis” triggers neoplastic transformation in surviving cells (Greenberg, 2005). This presents cells with a problem due to the need for unlimited proliferative capacity. Cancer cells bypass this problem by activating a telomere maintenance mechanism (TMM) that returns chromosome stability and allows continued proliferation.

I.2 Telomerase

Most cancer tumor types express the enzyme telomerase to allow for continued proliferation. It is composed of two subunits: the reverse transcriptase subunit of telomerase performs de novo synthesis of telomere repeats; the associated RNA molecule, TERC, acts as a template so that the reverse transcriptase can add the repeats to the end of the chromosome (Greider and Blackburn, 1985; Morin, 1989). The proliferative capacity that telomerase provides is not only important for cancer cells but also stem cells. Stem cells allow for tissue regeneration and require telomerase expression to prevent telomere shortening and therefore dysfunction, senescence and/or apoptosis (Batista, 2014). Limited telomerase activity in an adult organism cannot compensate for continued telomere shortening over a lifetime and explains the link between telomeres, telomerase and aging (Collins and Mitchell, 2002).

I.3 Alternative lengthening of telomeres

A minority of cancers use the recombination-based TMM of ALT for continual proliferation. Most of the cancer types that utilize ALT are of mesenchymal origin
4including osteosarcomas, soft-tissue sarcomas and the adult brain cancer glioblastoma multiforme. Among the most common cancer types such as breast carcinomas, ALT rarely plays a role (Bryan et al., 1997; Ferrandon et al., 2013; Hakin-Smith et al., 2003; Henson et al., 2005; Lafferty-Whyte et al., 2009; Subhawong et al., 2009;). Rare tumors express both telomerase and ALT in the same tumor (Gocha et al., 2013). Telomerasepositive tumors are capable of switching TMM to ALT when treated with telomerase inhibitors (Hu et al., 2012). These studies indicate that the need for ALT therapies may expand beyond that of ALT-positive tumors alone (Gocha et al., 2013; Hu et al., 2012).
One type of recombination that occurs in ALT cancer cells is homologous

recombination (HR)-dependent DNA replication telomere copying (Dunham et al.,

2000). A second type of recombination occurs as post-replicative exchanges between sister chromatids. A longer telomere on a sister chromatid can exchange a portion of its sequence with a shorter telomere on a sister chromatid to yield a net gain in length on the shorter sister chromatid. Intertelomeric exchanges could also take place between nearby homologous chromosomes (Conomos et al., 2014; Londoño-Vallejo et al., 2004). A third

type of recombination is termed HR-mediated t-loop junction resolution. The t-loop

conformation of the telomere may allow for intratelomeric recombination and resolution of the t-loop. A circular extrachromosomal telomere repeat substrate, called a t-circle, is produced from this type of recombination and can be used for rolling circle amplification (RCA) to rapidly add on to critically short telomeres. This may contribute to the heterogeneous lengths of telomeres in ALT cells (Cesare and Griffith, 2004; Henson et

al., 2009).

5
ALT cancer cells have distinct phenotypes that can be exploited to study proteins that affect ALT (Figure 2). ALT cancer cells have heterogeneous telomere lengths ranging from short telomeres, 2 kb in length, to long telomeres, up to 50 kb in length (Figure 2A). The long telomeres found in ALT cells are much longer than telomere lengths observed in telomerase-positive cells, which are approximately 10 kb in length (Bryan et al., 1995; Park et al., 1998). Another phenotype is the presence of ALT- associated promyelocytic leukemia protein (PML) bodies (APBs), a nuclear body shown by fluorescence in situ hybridization (FISH)/immunofluorescence (IF) studies to contain telomeric DNA, shelterin proteins, DNA repair proteins and PML (Yeager et al., 1999) (Figure 2B). An elevated number of telomere sister chromatid exchanges (T-SCEs), as compared to telomerase-positive cells and the numbers of SCEs found elsewhere in the genome, can also be observed in ALT cells (Betcher et al., 2004; Londono-Vallejo et al., 2004) (Figure 2C). A unique assay detects T-SCEs and is termed chromosomeorientation FISH (CO-FISH) in which G- and C-strands of DNA at the telomere are uniquely labeled to detect the exchange between sister chromatids (Bailey et al., 1996; Bailey et al., 2004; Goodwin and Meyne, 1993). ALT cells also have an abundance of linear and circular single-stranded or double-stranded extrachromosomal telomere repeat (ECTR) DNA, (telomeric DNA that has been detached from the chromosome), as compared to telomerase-positive cells (Nabetani and Ishikawa, 2009) (Figure 2D).

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    Table S2.Up or down regulated genes in Tcof1 knockdown neuroblastoma N1E-115 cells involved in differentbiological process analysed by DAVID database Pop Pop Fold Term PValue Genes Bonferroni Benjamini FDR Hits Total Enrichment GO:0044257~cellular protein catabolic 2.77E-10 MKRN1, PPP2R5C, VPRBP, MYLIP, CDC16, ERLEC1, MKRN2, CUL3, 537 13588 1.944851 8.64E-07 8.64E-07 5.02E-07 process ISG15, ATG7, PSENEN, LOC100046898, CDCA3, ANAPC1, ANAPC2, ANAPC5, SOCS3, ENC1, SOCS4, ASB8, DCUN1D1, PSMA6, SIAH1A, TRIM32, RNF138, GM12396, RNF20, USP17L5, FBXO11, RAD23B, NEDD8, UBE2V2, RFFL, CDC GO:0051603~proteolysis involved in 4.52E-10 MKRN1, PPP2R5C, VPRBP, MYLIP, CDC16, ERLEC1, MKRN2, CUL3, 534 13588 1.93519 1.41E-06 7.04E-07 8.18E-07 cellular protein catabolic process ISG15, ATG7, PSENEN, LOC100046898, CDCA3, ANAPC1, ANAPC2, ANAPC5, SOCS3, ENC1, SOCS4, ASB8, DCUN1D1, PSMA6, SIAH1A, TRIM32, RNF138, GM12396, RNF20, USP17L5, FBXO11, RAD23B, NEDD8, UBE2V2, RFFL, CDC GO:0044265~cellular macromolecule 6.09E-10 MKRN1, PPP2R5C, VPRBP, MYLIP, CDC16, ERLEC1, MKRN2, CUL3, 609 13588 1.859332 1.90E-06 6.32E-07 1.10E-06 catabolic process ISG15, RBM8A, ATG7, LOC100046898, PSENEN, CDCA3, ANAPC1, ANAPC2, ANAPC5, SOCS3, ENC1, SOCS4, ASB8, DCUN1D1, PSMA6, SIAH1A, TRIM32, RNF138, GM12396, RNF20, XRN2, USP17L5, FBXO11, RAD23B, UBE2V2, NED GO:0030163~protein catabolic process 1.81E-09 MKRN1, PPP2R5C, VPRBP, MYLIP, CDC16, ERLEC1, MKRN2, CUL3, 556 13588 1.87839 5.64E-06 1.41E-06 3.27E-06 ISG15, ATG7, PSENEN, LOC100046898, CDCA3, ANAPC1, ANAPC2, ANAPC5, SOCS3, ENC1, SOCS4,
  • “Molecular Characterisation of Helq Helicase's Role In

    “Molecular Characterisation of Helq Helicase's Role In

    “MOLECULAR CHARACTERISATION OF HELQ HELICASE’S ROLE IN DNA REPAIR AND GENOME STABILITY” Rafal Lolo University College London and Cancer Research UK London Research Institute PhD Supervisor: Simon Boulton A thesis submitted for the degree of Doctor of Philosophy University College London September 2018 Declaration I Rafal Lolo confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Abstract Maintenance of genome stability is a critical condition that ensures that daughter cells acquire an accurate copy of the genetic information from the parental cell. DNA replication stress that arises from blocked replication forks, can be a major challenge to genome integrity. Cells have therefore developed complex mechanisms to detect and deal with the replication-associated DNA damage. Intra-S-phase ATR checkpoint, FA pathway and RAD51 paralog BCDX2 complex together constitute key components of the replication stress response system that is essential to sense, repair and restart damaged forks. Previous studies in D. melanogaster and C. elegans have positioned HELQ as an important factor in DNA damage repair and maintaining genome stability. In this work I develop and combine biochemical assays, proteomic studies, mouse model and molecular biology tools to further characterise HELQ function in DNA repair and genome stability. I establish that HELQ plays a pivotal role in the replication stress response in mammalian cells. By developing a system in which I was able to pull down tagged HELQ and subject it to Mass Spectrometry analysis I identified its molecular partners and showed that HELQ interacts with, and interfaces between, the central FANCD2/FANCI heterodimer and the downstream RAD51 paralog BCDX2 complex.
  • Acetylation of BLM Protein Regulates Its Function in Response to DNA Damage Cite This: RSC Adv.,2017,7,55301 Yankun Wang and Jianyuan Luo *

    Acetylation of BLM Protein Regulates Its Function in Response to DNA Damage Cite This: RSC Adv.,2017,7,55301 Yankun Wang and Jianyuan Luo *

    RSC Advances View Article Online PAPER View Journal | View Issue Acetylation of BLM protein regulates its function in response to DNA damage Cite this: RSC Adv.,2017,7,55301 Yankun Wang and Jianyuan Luo * Bloom syndrome is an autosomal recessive disease with phenotypes of cancer predisposition and premature aging caused by mutations of the blm gene. BLM belongs to the RecQ DNA helicase family and functions in maintaining genomic stability. In this study, we found that several lysine residues of BLM were acetylated in cells. The dynamic acetylation levels of BLM were regulated by CBP/p300 and SIRT1. Received 15th June 2017 We further identified that five lysines, K476, K863, K1010, K1329, and K1411, are the major acetylation Accepted 29th November 2017 sites. Treating cells with different DNA damage agents found that acetylation of BLM was different in DOI: 10.1039/c7ra06666j response to etoposide and hydroxyurea, suggesting that BLM acetylation may have multiple functions in rsc.li/rsc-advances DNA repair. Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Introduction recombination and makes DNA back to integrated condition.14 On the other hand, BLM interacts with 53BP1 and completes Bloom syndrome protein (BLM), coded by the blm gene, is the repair in the NHEJ pathway.11 It has been found that BLM is a 1417 amino acid protein. Mutations or deletions of the blm sensitive to multiple stress factors, including hydroxyurea (HU), gene lead to Bloom Syndrome (BS).1 It is an inherited etoposide and ionizing radiation (IR) which all
  • Hyper Telomere Recombination Accelerates Replicative Senescence and May Promote Premature Aging

    Hyper Telomere Recombination Accelerates Replicative Senescence and May Promote Premature Aging

    Hyper telomere recombination accelerates replicative senescence and may promote premature aging R. Tanner Hagelstroma,b, Krastan B. Blagoevc,d, Laura J. Niedernhofere, Edwin H. Goodwinf, and Susan M. Baileya,1 aDepartment of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO 80523-1618; bPharmaceutical Genomics Division, Translational Genomics Research Institute, Phoenix, AZ 85004; cNational Science Foundation, Arlington, VA 22230; dDepartment of Physics Cavendish Laboratory, Cambridge University, Cambridge CB3 0HE, United Kingdom; eDepartment of Microbiology and Molecular Genetics, University of Pittsburgh, School of Medicine and Cancer Institute, Pittsburgh, PA 15261; and fKromaTiD Inc., Fort Collins, CO 80524 Edited* by José N. Onuchic, University of California San Diego, La Jolla, CA, and approved August 3, 2010 (received for review May 7, 2010) Werner syndrome and Bloom syndrome result from defects in the cell-cycle arrest known as senescence (12). Most human tissues lack RecQ helicases Werner (WRN) and Bloom (BLM), respectively, and sufficient telomerase activity to maintain telomere length through- display premature aging phenotypes. Similarly, XFE progeroid out life, limiting cell division potential. The majority of cancers syndrome results from defects in the ERCC1-XPF DNA repair endo- circumvent this tumor-suppressor mechanism by reactivating telo- nuclease. To gain insight into the origin of cellular senescence and merase (13), thus removing telomere shortening as a barrier to human aging, we analyzed the dependence of sister chromatid continuous proliferation. In some situations, a recombination- exchange (SCE) frequencies on location [i.e., genomic (G-SCE) vs. telo- based mechanism known as “alternative lengthening of telomeres” meric (T-SCE) DNA] in primary human fibroblasts deficient in WRN, (ALT) maintains telomere length in the absence of telomerase (14).
  • Understanding Nucleotide Excision Repair and Its Roles in Cancer and Ageing

    Understanding Nucleotide Excision Repair and Its Roles in Cancer and Ageing

    REVIEWS DNA DAMAGE Understanding nucleotide excision repair and its roles in cancer and ageing Jurgen A. Marteijn*, Hannes Lans*, Wim Vermeulen, Jan H. J. Hoeijmakers Abstract | Nucleotide excision repair (NER) eliminates various structurally unrelated DNA lesions by a multiwise ‘cut and patch’-type reaction. The global genome NER (GG‑NER) subpathway prevents mutagenesis by probing the genome for helix-distorting lesions, whereas transcription-coupled NER (TC‑NER) removes transcription-blocking lesions to permit unperturbed gene expression, thereby preventing cell death. Consequently, defects in GG‑NER result in cancer predisposition, whereas defects in TC‑NER cause a variety of diseases ranging from ultraviolet radiation‑sensitive syndrome to severe premature ageing conditions such as Cockayne syndrome. Recent studies have uncovered new aspects of DNA-damage detection by NER, how NER is regulated by extensive post-translational modifications, and the dynamic chromatin interactions that control its efficiency. Based on these findings, a mechanistic model is proposed that explains the complex genotype–phenotype correlations of transcription-coupled repair disorders. The integrity of DNA is constantly threatened by endo­ of an intricate DNA-damage response (DDR), which genously formed metabolic products and by-products, comprises sophisticated repair and damage signalling such as reactive oxygen species (ROS) and alkylating processes. The DDR involves DNA-damage sensors and agents, and by its intrinsic chemical instability (for exam­ signalling kinases that regulate a range of downstream ple, by its ability to spontaneously undergo hydrolytic mediator and effector molecules that control repair, cell deamination and depurination). Environmental chemi­ cycle progression and cell fate4. The core of this DDR is cals and radiation also affect the physical constitution of formed by a network of complementary DNA repair sys­ DNA1.
  • University of Cincinnati

    University of Cincinnati

    UNIVERSITY OF CINCINNATI _____________ , 20 _____ I,______________________________________________, hereby submit this as part of the requirements for the degree of: ________________________________________________ in: ________________________________________________ It is entitled: ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ Approved by: ________________________ ________________________ ________________________ ________________________ ________________________ BLM is a Suppressor of DNA Recombination A dissertation submitted to the Division of Research and Advanced Studies Of the University of Cincinnati In partial fulfillment of the Requirements for the degree of Doctorate of Philosophy (Ph.D.) In the Department of Molecular Genetics, Microbiology, and Biochemistry Of the College of Arts and Sciences 2002 by Joel E. Straughen B.S., The Ohio State University, 1985 M.D., University of Cincinnati, 2002 Committee Chair: Joanna Groden, Ph.D. i ABSTRACT Bloom’s syndrome (BS) is a rare, recessive chromosome breakage disorder characterized by small stature, sun sensitivity, facial erythema, immunodeficiency, female subfertility, male infertility, and a predisposition to a variety of cancers. When this body of work was started, the gene for Bloom’s syndrome (BLM)hadyettobe identified. This work presents characterization of the genomic region at BLM and the identification of BLM. With the cloning