DOCSLIB.ORG

The Highly Parallel Homology Directed Repair Assay and the Analysis of BRCA1 Variants

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
The Highly Parallel Homology Directed Repair Assay and the Analysis of BRCA1 Variants The Highly Parallel Homology Directed Repair Assay and the Analysis of BRCA1 Variants Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Muhtadi Muhammad Islam, B.E. Biomedical Sciences Graduate Program The Ohio State University 2016 Dissertation Committee: Jeffrey Parvin, MD, PhD, Advisor Michael Freitas, PhD Kun Huang, PhD Amanda Toland, PhD Copyrighted by Muhtadi Muhammad Islam 2016 Abstract DNA damage and repair are processes that have been linked to cancer, both in carcinogenesis and in the treatment of cancer. We explore two different DNA repair factors, BRCA1 and HDAC10, in two distinct studies. BRCA1 is a highly penetrant gene in both breast and ovarian cancer and with several functions. We analyze BRCA1 in a novel assay that scores the homologous recombination repair function of several thousand BRCA1 variants in parallel. Our goal is to create a structure function map of BRCA1 at the amino acid resolution to not only prove the novel highly parallel functional assay, but also to provide clinically relevant data of BRCA1 variants with respect to homologous recombination repair. HDAC10 is a histone deacetylase with only one known function, stimulating homologous recombination repair of double strand breaks. Utilizing genome databases, we found that HDAC10 is deleted in up to 10% of ovarian tumors. We also found that low HDAC10 expression correlated with platinum therapy sensitivity. We demonstrated general HDAC inhibition enhanced cisplatin therapy in primary ovarian carcinomas. Additionally, HDAC10 specific inhibition sensitized a BRCA1 deficient ovarian cancer cell line to both DNA damage and cisplatin therapy. We suggest that HDAC10 is an important agent in the mechanisms of general HDAC inhibition as well as a potential new target for BRCA1 deficient ovarian tumors. ii Dedication This document is dedicated to the people and families who are struggling with cancer. iii Acknowledgements The primary driving force who supported me throughout my research was Dr. Jeffrey Parvin. His support was unwavering throughout the years, despite the few triumphs amongst the many setbacks. My committee has provided me with invaluable feedback and regularly reinvigorated my drive through their excitement at the potential of my work. My colleagues have been a continual source of both technical and emotional support, something I will dearly miss. My parents and sisters have been remarkably patient with me during these years and always believed in my ability even when I had my doubts. Finally, to my wife, who has been my partner in all my trials and tribulations these last few years. Andria has done so much more than simply share my burdens, she transformed them time and again so they were less heavy for both of us. This is something I am grateful for beyond words. Thank you all. iv Vita May 2003 .......................................................Centerville High School April 2007 .......................................................B.E. Chemical Engineering University of Dayton August 2007-present .....................................Graduate Research Assistant Department of Biomedical Informatics The Ohio State University Publications Zhang, J., Lu, K., Xiang, Y., Islam, M., Kotian, S., Kais, Z., ... & Huang, K. (2012). Weighted frequent gene co-expression network mining to identify genes involved in genome stability. PLoS Comput Biol, 8(8), e1002656. Starita, L. M., Young, D. L., Islam, M., Kitzman, J. O., Gullingsrud, J., Hause, R. J., ... & Fields, S. (2015). Massively parallel functional analysis of BRCA1 RING domain variants. Genetics, 200(2), 413-422. Field of Study Major Field: Biomedical Sciences v Table of Contents Abstract ...............................................................................................................................ii Dedication .......................................................................................................................... iii Acknowledgements ............................................................................................................ iv Vita ...................................................................................................................................... v List of Figures ..................................................................................................................... ix List of Tables....................................................................................................................... xi Chapter 1: A Review of DNA Repair and Cancer ................................................................ 1 1.1 A Recent History of Molecular Biology ................................................................ 2 1.2 BRCA1 and Breast Cancer ..................................................................................... 4 1.3 The link between DNA damage and cancer ......................................................... 7 1.4 DNA Repair review ............................................................................................... 9 1.4a Non-homologous End Joining ......................................................................... 14 1.4b Homologous Recombination .......................................................................... 15 1.5 BRCA1 ................................................................................................................. 17 1.6 Cancer treatment ............................................................................................... 20 1.7 Summary of studies presented .......................................................................... 23 Chapter 2: Highly Parallel Homology Directed Repair ..................................................... 25 2.1 Abstract .............................................................................................................. 26 vi 2.2 Introduction........................................................................................................ 26 2.3 Materials and Methods ...................................................................................... 28 2.3a Homology Directed Repair Assay ................................................................... 28 2.3b HeLa DR-FRT generation ................................................................................. 30 2.3c HeLa BRCA1-variant cell line generation ........................................................ 34 2.3d Highly Parallel Homology Directed Repair Assay ........................................... 35 2.3e Construction of barcoded variant libraries of BRCA1 .................................... 36 2.3f Sequencing to link the BRCA1 N-terminal variants to their barcode ............. 37 2.3g Sequencing barcodes from genomic DNA ...................................................... 38 2.3h Determining a functional score for each BRCA1 variant ................................ 39 2.3i Statistics .......................................................................................................... 42 2.4 Validation Results ............................................................................................... 42 2.5 BRCA1 variants ................................................................................................... 51 2.6 Discussion ........................................................................................................... 57 Chapter 3: HDAC10 as a potential therapeutic target in ovarian cancer ........................ 61 3.1 Abstract .............................................................................................................. 62 3.2 Introduction........................................................................................................ 62 3.3 Materials and Methods ...................................................................................... 64 3.3a Cell Culture and Reagents .............................................................................. 64 3.3b Homology Directed Repair Assay (HDR) ......................................................... 65 3.3c Comet Assay ................................................................................................... 65 vii 3.3d MTT Assay ....................................................................................................... 67 3.3e Statistics .......................................................................................................... 67 3.4 Results ................................................................................................................ 68 3.5 Discussion ........................................................................................................... 80 3.6 Tables ................................................................................................................. 85 Chapter 4: Summary and Future Directions .................................................................... 86 4.1 DNA repair and cancer ....................................................................................... 87 4.2 BRCA1 ................................................................................................................. 88 4.2a Highly Parallel Homology Directed Repair ..................................................... 88 4.2b BRCA1 Future Directions ................................................................................ 89 4.3 HDAC10 .............................................................................................................
Load more

Recommended publications
  • Characterization of the Interplay Between DNA Repair and CRISPR/Cas9-Induced DNA Lesions
    Characterization of the interplay between DNA repair and CRISPR/Cas9-induced DNA lesions Anne Bothmer, Tanushree Phadke, Luis Barrera, Carrie Margulies, Hari Jayaram, Vic Myer, Cecilia Cotta-Ramusino Editas Medicine • 11 Hurley Street • Cambridge, MA 02141 Background Mechanism of generation of insertions differs between D10A and N863A Conclusions 1. WT-Cas9 and Cas9 paired nickases led to the activation of double-strand The CRISPR/Cas9 system provides a break (DSB) response pathways at similar rates and the presence and versatile toolkit for genome engineering that polarity of the overhang structure is a determinant of DSB-repair pathway can introduce a variety of DNA lesions at choice. specific genomic locations. However, a better understanding of the exact nature of these lesions and the repair pathways engaged as a consequence thereof is critical to realizing the basic research and therapeutic potential of this technology.. Here we characterize the DNA structures arising from the use of Cas9 variants directed to the endogenous human beta-globin locus. The different lesions arising from each Cas9 variant resulted in Blunt ends generated by Wt 5’ protruding ends generated 3’ protruding ends generated by the engagement of different endogenous Microhomology Microhomology Cas9 are repaired mostly by by D10A paired nickases are N863A pair nickases are canonical Non Homologous predominantly repaired by predominantly repaired by repair pathways. End Joining (c-NHEJ) Homology Directed Repair alternative Non Homologous (HDR) End Joining (a-NHEJ) Schematic of Cas9 variants Different donors engage different repair pathways 2. The nature of the donor is an important determinant in repair pathway engagement regardless of the lesion generated: ssDNA Donor engages Single dsDNA Donors engage Stranded Template Repair Homologous Recombination (Homologous Recombination- repair Gene Conversion Gene Gene Correction Gene Gene Conversion Gene independent) Neg.
    [Show full text]
  • Large XPF-Dependent Deletions Following Misrepair of a DNA Double Strand Break Are Prevented by the RNA:DNA Helicase Senataxin
    www.nature.com/scientificreports OPEN Large XPF-dependent deletions following misrepair of a DNA double strand break are prevented Received: 26 October 2017 Accepted: 9 February 2018 by the RNA:DNA helicase Published: xx xx xxxx Senataxin Julien Brustel1, Zuzanna Kozik1, Natalia Gromak2, Velibor Savic3,4 & Steve M. M. Sweet1,5 Deletions and chromosome re-arrangements are common features of cancer cells. We have established a new two-component system reporting on epigenetic silencing or deletion of an actively transcribed gene adjacent to a double-strand break (DSB). Unexpectedly, we fnd that a targeted DSB results in a minority (<10%) misrepair event of kilobase deletions encompassing the DSB site and transcribed gene. Deletions are reduced upon RNaseH1 over-expression and increased after knockdown of the DNA:RNA helicase Senataxin, implicating a role for DNA:RNA hybrids. We further demonstrate that the majority of these large deletions are dependent on the 3′ fap endonuclease XPF. DNA:RNA hybrids were detected by DNA:RNA immunoprecipitation in our system after DSB generation. These hybrids were reduced by RNaseH1 over-expression and increased by Senataxin knock-down, consistent with a role in deletions. Overall, these data are consistent with DNA:RNA hybrid generation at the site of a DSB, mis-processing of which results in genome instability in the form of large deletions. DNA is the target of numerous genotoxic attacks that result in diferent types of damage. DNA double-strand breaks (DSBs) occur at low frequency, compared with single-strand breaks and other forms of DNA damage1, however DSBs pose the risk of translocations and deletions and their repair is therefore essential to cell integrity.
    [Show full text]
  • Functional Connection Between Rad51 and PML in Homology-Directed Repair
    Functional Connection between Rad51 and PML in Homology-Directed Repair Sergei Boichuk1, Liang Hu1¤a, Kathleen Makielski1, Pier Paolo Pandolfi2, Ole V. Gjoerup1*¤b 1 Cancer Virology Program, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania, United States of America, 2 Department of Medicine, Beth Israel Deaconess Cancer Center, Harvard Medical School, Boston, Massachusetts, United States of America Abstract The promyelocytic leukemia protein (PML) is a tumor suppressor critical for formation of nuclear bodies (NBs) performing important functions in transcription, apoptosis, DNA repair and antiviral responses. Earlier studies demonstrated that simian virus 40 (SV40) initiates replication near PML NBs. Here we show that PML knockdown inhibits viral replication in vivo, thus indicating a positive role of PML early in infection. SV40 large T antigen (LT) induces DNA damage and, consequently, nuclear foci of the key homologous recombination repair protein Rad51 that colocalize with PML. PML depletion abrogates LT-induced Rad51 foci. LT may target PML NBs to gain access to DNA repair factors like Rad51 that are required for viral replication. We have used the SV40 model to gain insight to DNA repair events involving PML. Strikingly, even in normal cells devoid of viral oncoproteins, PML is found to be instrumental for foci of Rad51, Mre11 and BRCA1, as well as homology- directed repair after double-strand break (DSB) induction. Following LT expression or external DNA damage, PML associates with Rad51. PML depletion also causes a loss of RPA foci following c-irradiation, suggesting that PML is required for processing of DSBs. Immunofluorescent detection of incorporated BrdU without prior denaturation indicates a failure to generate ssDNA foci in PML knockdown cells upon c-irradiation.
    [Show full text]
  • Functions of BRCA1, 53BP1 and SUMO Isoforms in DNA Double-Strand Break Repair in Mammalian Cells
    Functions of BRCA1, 53BP1 and SUMO isoforms in DNA double-strand break repair in mammalian cells DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Yiheng Hu Graduate Program in Molecular, Cellular and Developmental Biology The Ohio State University 2014 Dissertation Committee: Dr. Jeffrey Parvin, Advisor Dr. Altaf Wani Dr. Qianben Wang Dr. Robin Wharton Copyright by Yiheng Hu 2014 Abstract In this dissertation study, we have investigated the protein functions in DNA double-strand break (DSB) repair of three important factors, BRCA1, 53BP1 and SUMO isoforms, at levels of biochemical activity, protein dynamics and chromosomal DNA repair. Our work reveals novel mechanisms of these proteins functioning in response to DSB damage, hence providing insights of where and how they are actively involved in each subpathway of DSB repair. In the first part of our work, we studied BRCA1, a tumor suppressor important for the maintenance of genomic stability including centrosome control and DSB repair, and found that a putative enzymatic mutant of BRCA1— BRCA1(I26A), which had been thought to disrupt its E3 ligase activity, was still functional in the cellular processes of regulating centrosome number and homologous recombination-dependent DSB repair, thereby raising a question of whether I26A mutant is indeed inert. Reevaluation of the ubiquitination activity of this BRCA1(I26A) mutant revealed that it is an active E3 ubiquitin ligase when associated with the appropriate E2 factor. We then think that conclusions about the dispensability of the BRCA1-dependent enzymatic activity in various cellular processes should be reconsidered.
    [Show full text]
  • Comparative Analysis of NHEJ and HDR Repair Pathways for Genomic Editing Using CRISPR/Cas9 Technology in ES Cells
    Comparative analysis of NHEJ and HDR repair pathways for genomic editing using CRISPR/Cas9 technology in ES cells Astrid Jensen1, Jeroen DeGroot1, Uma Saha2, Steve Festin2, Anne-Marie Zuurmond1 1Charles River Leiden, Darwinweg 24, 2333 CR Leiden, Netherlands; 2Charles River Wilmington, 251 Ballardvale Street, Wilmington MA 01887, USA 1 Introduction 2 Double-strand break repair pathways Cas9 Cut A * The error-prone non-homology end joining (NHEJ) repair pathway is often the pathway of choice when utilizing 5’ Insertion site 3’ Cut CRISPR/Cas9 to generate a genetic knockout. Despite the high efficiency of creating indel mutations, the pathway has its * drawbacks. Deletions are often in-frame, which might result in semi-functional proteins. Indels can easily span from 1-200 gRNA bp, without any control on their exact length or location (upstream/downstream) which can make indel identification and 5’ validation an expensive and time consuming task. Double 5’ stranded break (DSB) B C The homology driven repair (HDR) pathway allows for precise editing of the genome by including a repair template in the NHEJ HDR 5’ process. Using a single-stranded template ensures that this template is not randomly inserted elsewhere in the genome or 5’ SNP 5’ 5’ is targeted by the Cas9 enzyme. The introduced sequence also gives the opportunity for fast and efficient PCR validation of * edited clones prior to sequencing. Here we performed a comparative analysis of the two repair mechanisms to investigate Indel SNP whether the HDR pathway is a good alternative to the NHEJ pathway for high frequency generation of genetic knockouts in 5’ 5’ 5’ * 5’ mouse C57BL/6NCrl embryonic stem cells (ESC), but with the concomitant advantage of precise genome editing.
    [Show full text]
  • Recruitment of Mismatch Repair Proteins to the Site of DNA Damage in Human Cells
    3146 Research Article Recruitment of mismatch repair proteins to the site of DNA damage in human cells Zehui Hong, Jie Jiang, Kazunari Hashiguchi, Mikiko Hoshi, Li Lan and Akira Yasui* Department of Molecular Genetics, Institute of Development, Aging and Cancer, Tohoku University, Seiryomachi 4-1, Aobaku, Sendai 980-8575, Japan *Author for correspondence (e-mail: [email protected]) Accepted 8 July 2008 Journal of Cell Science 121, 3146-3154 Published by The Company of Biologists 2008 doi:10.1242/jcs.026393 Summary Mismatch repair (MMR) proteins contribute to genome stability the PCNA-binding domain of MSH6. MSH2 is recruited to the by excising DNA mismatches introduced by DNA polymerase. DNA damage site through interactions with either MSH3 or Although MMR proteins are also known to influence cellular MSH6, and is required for recruitment of MLH1 to the damage responses to DNA damage, how MMR proteins respond to DNA site. We found, furthermore, that MutSβ is also recruited to damage within the cell remains unknown. Here, we show that UV-irradiated sites in nucleotide-excision-repair- and PCNA- MMR proteins are recruited immediately to the sites of various dependent manners. Thus, MMR and its proteins function not types of DNA damage in human cells. MMR proteins are only in replication but also in DNA repair. recruited to single-strand breaks in a poly(ADP-ribose)- dependent manner as well as to double-strand breaks. Using Supplementary material available online at mutant cells, RNA interference and expression of fluorescence- http://jcs.biologists.org/cgi/content/full/121/19/3146/DC1
    [Show full text]
  • The Role of Sgs1 and Exo1 in the Maintenance of Genome Stability. Lillian Campos-Doerfler University of South Florida, [email protected]
    University of South Florida Scholar Commons Graduate Theses and Dissertations Graduate School November 2017 The Role of Sgs1 and Exo1 in the Maintenance of Genome Stability. Lillian Campos-Doerfler University of South Florida, [email protected] Follow this and additional works at: http://scholarcommons.usf.edu/etd Part of the Cell Biology Commons, and the Molecular Biology Commons Scholar Commons Citation Campos-Doerfler, Lillian, "The Role of Sgs1 and Exo1 in the Maintenance of Genome Stability." (2017). Graduate Theses and Dissertations. http://scholarcommons.usf.edu/etd/7006 This Dissertation is brought to you for free and open access by the Graduate School at Scholar Commons. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. The Role of Sgs1 and Exo1 in the Maintenance of Genome Stability By Lillian Campos-Doerfler A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy With a concentration in Cell and Molecular Biology Department of Cell Biology, Microbiology and Molecular Biology College of Arts and Sciences University of South Florida Major Professor: Kristina Schmidt, Ph.D. Meera Nanjundan, Ph.D. Stanley Stevens, Ph.D. Sandy Westerheide, Ph.D. Date of Approval: November 17, 2017 Keywords: Genome Instability, DNA repair, RecQ Helicases, Sgs1, Exo1 Copyright 2017, Lillian Campos-Doerfler Dedication I would like to dedicate this dissertation to my amazing and supportive family. To my husband Eric for his constant encouragement and patience, my sister Connie for her motivational phone calls and guidance, and my parents Wanda and Jaime for everything they have done that allowed for me to pursue my dream.
    [Show full text]
  • RPN530 DNA Repair Slides with Notes 2014.Pptx
    DNA$Repair$and$Cancer$ RPN$530$Oncology$for$Scien4sts$ Lecture$ Thomas$Melendy,$PhD$ Depts:$Microbiology$&$Immunology,$ and$Biochemistry,$UB$SOM$ Office:$213$BRB,$UB$SOM$ TMelendy@buffalo.edu$ DNA$Repair$and$Cancer$ Suggested$addi4onal$resources:$ Weinberg$RA$(2007)$The$Biology$of$Cancer.$ Garland$Science,$New$York,$(Chapter$12).$$ Friedberg$EC,$Walker$GC,$Siede$W,$Wood$ RD,$Shultz$RA,$Ellenberger$T$(2006)$DNA$ Repair$and$Mutagenesis,$ASM$Press,$ Washington,$DC.$$ DNA$Repair$and$Cancer$ RNA$and$proteins$can$be$totally$degraded$when$ damaged,$and$re\synthesiZed$when$needed$ DNA$is$not$totally$degraded$when$damaged$ $ \$repaired$at$a$nucleo4de$level$or$in$small$patches$ DNA$repair$maintains$integrity$of$the$gene4c$ informa4on$ $ $\$DNA$\>$RNA$\>$protein$ Importance$of$DNA$Repair$ All$organisms,$from$bacteria$to$yeast$to$humans,$have$ mul4ple$DNA$repair$mechanisms$and$pathways$ Many$of$the$genes,$proteins$and$repair$pathways$are$ evolu4onarily$conserved$ ~$150$human$genes$encode$DNA$repair$proteins,$and$ many$more$involved$in$DNA$damage$response$(DDR)$ Defects$in$DNA$Repair$can$cause$Cancer$ DNA$Repair$ Types$of$DNA$damage$ Types$of$DNA$repair$pathways$ DNA$damage,$repair,$and$cancer$ DNA$Repair$ Types$of$DNA$damage$ Errors$in$DNA$synthesis$ Spontaneous/endogenous$sources$ Exogenous$sources$ Replica4on$Errors$in$the$Genome$ DNA$polymerase$delta$(δ)$copies$DNA$with$ high$fidelity$(proofreading$mutant$D400A)$ \$Low$error$rate$in$copying:$~1$error/105$bp$ Human$genome$has$~6x109$bp$ \$6x109$/$1x105$=$~60,000$errors!$ How$does$the$cell$prevent/correct$these$
    [Show full text]
  • Silencing of End-Joining Repair for Efficient Site-Specific Gene Insertion After TALEN/CRISPR Mutagenesis in Aedes Aegypti
    Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis in Aedes aegypti Sanjay Basua,1, Azadeh Aryana,1, Justin M. Overcasha, Glady Hazitha Samuela, Michelle A. E. Andersona, Timothy J. Dahlemb, Kevin M. Mylesa,2, and Zach N. Adelmana,2 aFralin Life Science Institute and Department of Entomology, Virginia Tech, Blacksburg, VA 24061; and bMutation Generation and Detection Core, Health Science Center Core Research Facility, University of Utah, Salt Lake City, UT 84132 Edited by Anthony A. James, University of California, Irvine, CA, and approved February 24, 2015 (received for review February 6, 2015) Conventional control strategies for mosquito-borne pathogens range of potential target sequences, already leading to the pro- such as malaria and dengue are now being complemented by the posal of alternative gene drive designs and regulatory structure development of transgenic mosquito strains reprogrammed to based on these more facile genetic tools (5, 6). Identified in type generate beneficial phenotypes such as conditional sterility II Streptococcus pyogenes as a heritable immune defense mech- or pathogen resistance. The widespread success of site-specific anism against viruses and plasmids (7), endogenous CRISPR loci nucleases such as transcription activator-like effector nucleases are comprised of 30–40 nucleotide (nt) variable sequences sep- (TALENs) and clustered regularly interspaced short palindromic arated by short direct repeats. CRISPR locus transcripts are used repeats (CRISPR)/Cas9 in model organisms also suggests that re- to generate short CRISPR RNAs (crRNAs) that act in con- programmable gene drive systems based on these nucleases may junction with a common transactivating-RNA (trRNA) to guide be capable of spreading such beneficial phenotypes in wild mos- Cas9 endonuclease to the complementary double-stranded DNA quito populations.
    [Show full text]
  • Repair of DNA Interstrand Cross-Links: Interactions Between Homology-Dependent and Homology-Independent Pathways
    dna repair 5 (2006) 566–574 available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/dnarepair Repair of DNA interstrand cross-links: Interactions between homology-dependent and homology-independent pathways Huyong Zheng a,b, Xin Wang a, Randy J. Legerski c, Peter M. Glazer d, Lei Li a,c,∗ a Department of Experimental Radiation Oncology, Unit 66, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, United States b Capital University of Medical Science, Beijing Children’s Hospital, Beijing, China c Department of Molecular Genetics, The University of Texas, M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, United States d Department of Therapeutic Radiology, Yale University, School of Medicine, New Heaven, CT 06520, United States article info abstract Article history: DNA interstrand cross-links (ICLs) are complex DNA lesions generated by bifunctional alky- Received 21 October 2005 lating agents, a class of compounds extensively used in cancer chemotherapy. Formation of Received in revised form 29 an ICL covalently links the opposing strands of the double helix and results in severe dis- December 2005 ruptions of normal DNA functions, such as replication, transcription, and recombination. Accepted 16 January 2006 Because of the structural complexity, ICLs are most likely recognized by a variety of repair Published on line 29 March 2006 recognition proteins and processed through multiple mechanisms. To study the involve- ment of different repair pathways in ICL processing, we examined a variety of mammalian Keywords: mutants with distinct DNA repair deficiencies. We found that the presence of ICLs induces DNA frequent recombination between direct repeat sequences, suggesting that the single-strand Damage annealing pathway may be an important mechanism for the removal of ICLs situated Repair within direct repeats.
    [Show full text]
  • Mechanism of Homologous Recombination: Mediators and Helicases Take on Regulatory Functions
    REVIEWS Mechanism of homologous recombination: mediators and helicases take on regulatory functions Patrick Sung* and Hannah Klein‡ Abstract | Homologous recombination (HR) is an important mechanism for the repair of damaged chromosomes, for preventing the demise of damaged replication forks, and for several other aspects of chromosome maintenance. As such, HR is indispensable for genome integrity, but it must be regulated to avoid deleterious events. Mutations in the tumour- suppressor protein BRCA2, which has a mediator function in HR, lead to cancer formation. DNA helicases, such as Bloom’s syndrome protein (BLM), regulate HR at several levels, in attenuating unwanted HR events and in determining the outcome of HR. Defects in BLM are also associated with the cancer phenotype. The past several years have witnessed dramatic advances in our understanding of the mechanism and regulation of HR. Homologous recombination (HR) occurs in all life homologous chromosomes and generates chromosome- Meiosis I The successful completion of forms. HR studies were initially the domain of a few arm crossovers. These crossovers are essential for proper meiosis requires two cell aficionados who had a desire to understand how genetic chromosome segregation at the first meiotic division2,4. divisions. Meiosis I refers to the information is transferred and exchanged between Mitotic HR differs from meiotic HR in that few events are first division in which the pairs chromosomes. The isolation and characterization of associated with crossover and indeed, crossover formation of homologous chromosomes 1 DNA helicases are segregated into the two relevant mutants in Escherichia coli , and later in the is actively suppressed by specialized (see daughter cells.
    [Show full text]
  • A Consensus Model of Homology-Directed Repair Initiated by CRISPR/Cas Activity
    International Journal of Molecular Sciences Review A Consensus Model of Homology-Directed Repair Initiated by CRISPR/Cas Activity Kevin Bloh 1,2 and Natalia Rivera-Torres 1,* 1 Gene Editing Institute, Helen F. Graham Cancer Center & Research Institute, ChristianaCare, 4701 Ogletown-Stanton Road, Newark, DE 19710, USA; [email protected] 2 Department of Medical and Molecular Sciences, University of Delaware, Newark, DE 19710, USA * Correspondence: [email protected]; Tel.: +1-302-623-4754 Abstract: The mechanism of action of ssODN-directed gene editing has been a topic of discussion within the field of CRISPR gene editing since its inception. Multiple comparable, but distinct, path- ways have been discovered for DNA repair both with and without a repair template oligonucleotide. We have previously described the ExACT pathway for oligo-driven DNA repair, which consisted of a two-step DNA synthesis-driven repair catalyzed by the simultaneous binding of the repair oligonucleotide (ssODN) upstream and downstream of the double-strand break. In order to better elucidate the mechanism of ExACT-based repair, we have challenged the assumptions of the path- way with those outlines in other similar non-ssODN-based DNA repair mechanisms. This more comprehensive iteration of the ExACT pathway better described the many different ways where DNA repair can occur in the presence of a repair oligonucleotide after CRISPR cleavage, as well as how these previously distinct pathways can overlap and lead to even more unique repair outcomes. Keywords: CRISPR/Cas; homology directed repair; DNA repair; synthesis-dependent strand annealing; gene repair Citation: Bloh, K.; Rivera-Torres, N. A Consensus Model of Homology-Directed Repair Initiated by CRISPR/Cas Activity.
    [Show full text]