Publications by authors named "Maria Jasin"

155 Publications

Long-term survival of an ovarian cancer patient harboring a RAD51C missense mutation.

Cold Spring Harb Mol Case Stud 2021 Apr 8;7(2). Epub 2021 Apr 8.

Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York 10021, USA.

Mutations in homologous recombination (HR) genes predispose to cancer but also sensitize to chemotherapeutics. Although therapy can initially be effective, cancers frequently cease responding, leading to recurrence and poor prognosis. Here we identify a germline mutation in , a critical HR factor and known tumor suppressor, in an ovarian cancer patient with exceptionally long, progression-free survival. The RAD51C-T132P mutation is in a highly conserved residue within the nucleotide-binding site and interferes with single-strand DNA binding of the RAD51 paralog complex RAD51B-RAD51C-RAD51D-XRCC2 and association with another RAD51 paralog XRCC3. These biochemical defects lead to highly defective HR and drug sensitivity in tumor cells, ascribing RAD51C-T132P as a deleterious mutation that was likely causal for tumor formation. Conversely, its position within a critical site suggests that it is refractory to secondary mutations that would restore gene function and lead to therapy resistance. A need for a greater understanding of the relationship between mutation position and reversion potential of HR genes is underscored, as it may help predict the effectiveness of therapies in patients with HR-deficient cancers.
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http://dx.doi.org/10.1101/mcs.a006083DOI Listing
April 2021

Testicular Germ Cell Tumors Acquire Cisplatin Resistance by Rebalancing the Usage of DNA Repair Pathways.

Cancers (Basel) 2021 Feb 13;13(4). Epub 2021 Feb 13.

Department of Biomedicine and Prevention, University of Rome Tor Vergata, 00133 Rome, Italy.

Despite germ cell tumors (GCTs) responding to cisplatin-based chemotherapy at a high rate, a subset of patients does not respond to treatment and have significantly worse prognosis. The biological mechanisms underlying the resistance remain unknown. In this study, by using two TGCT cell lines that have acquired cisplatin resistance after chronic exposure to the drug, we identified some key proteins and mechanisms of acquired resistance. We show that cisplatin-resistant cell lines had a non-homologous end-joining (NHEJ)-less phenotype. This correlated with a reduced basal expression of TP53-binding protein 1 (53BP1) and DNA-dependent protein kinase (DNA-PKcs) proteins and reduced formation of 53BP1 foci after cisplatin treatment. Consistent with these observations, modulation of 53BP1 protein expression altered the cell line's resistance to cisplatin, and inhibition of DNA-PKcs activity antagonized cisplatin cytotoxicity. Dampening of NHEJ was accompanied by a functional increase in the repair of DNA double-strand breaks (DSBs) by the homologous recombination repair pathway. As a result, cisplatin-resistant cells were more resistant to PARP inhibitor (PARPi) monotherapy. Moreover, when PARPi was given in combination with cisplatin, it exerted an additive/synergistic effect, and reduced the cisplatin dose for cytotoxicity. These results suggest that treatment of cisplatin-refractory patients may benefit from low-dose cisplatin therapy combined with PARPi.
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http://dx.doi.org/10.3390/cancers13040787DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7917736PMC
February 2021

Kathryn Anderson (1952-2020).

Cell 2021 Mar;184(5):1123-1126

Developmental Biology Program, Sloan Kettering Institute, New York, NY 10065, USA. Electronic address:

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http://dx.doi.org/10.1016/j.cell.2021.01.054DOI Listing
March 2021

Allele-Specific Chromosome Removal after Cas9 Cleavage in Human Embryos.

Cell 2020 Dec 29;183(6):1650-1664.e15. Epub 2020 Oct 29.

Department of Pediatrics and Naomi Berrie Diabetes Center, Columbia University, New York, NY 10032, USA; Columbia University Stem Cell Initiative, New York, NY 10032, USA; Department of Obstetrics and Gynecology, Columbia University, New York, NY 10032, USA. Electronic address:

Correction of disease-causing mutations in human embryos holds the potential to reduce the burden of inherited genetic disorders and improve fertility treatments for couples with disease-causing mutations in lieu of embryo selection. Here, we evaluate repair outcomes of a Cas9-induced double-strand break (DSB) introduced on the paternal chromosome at the EYS locus, which carries a frameshift mutation causing blindness. We show that the most common repair outcome is microhomology-mediated end joining, which occurs during the first cell cycle in the zygote, leading to embryos with non-mosaic restoration of the reading frame. Notably, about half of the breaks remain unrepaired, resulting in an undetectable paternal allele and, after mitosis, loss of one or both chromosomal arms. Correspondingly, Cas9 off-target cleavage results in chromosomal losses and hemizygous indels because of cleavage of both alleles. These results demonstrate the ability to manipulate chromosome content and reveal significant challenges for mutation correction in human embryos.
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http://dx.doi.org/10.1016/j.cell.2020.10.025DOI Listing
December 2020

Assessing Homologous Recombination and Interstrand Cross-Link Repair in Embryonal Carcinoma Testicular Germ Cell Tumor Cell Lines.

Methods Mol Biol 2021 ;2195:113-123

Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy.

Testicular germ cell tumors (TGCTs) are typically exquisitely sensitive to DNA interstrand cross-link (ICLs) agents. ICLs covalently link both strands of the DNA duplex, impeding fundamental cellular processes like DNA replication to cause cell death. A leading drug used for the treatment of TGCTs is cisplatin, which introduces ICLs and leads to formation of double strand breaks (DSBs), a DNA lesion that can be repaired in the S/G2 phases of the cell cycle by homologous recombination (HR, also termed homology-direct repair). Although most TGCTs respond to cisplatin-induced ICLs, a fraction is resistant to treatment. One proposed mechanism of TGCT resistance to cisplatin is an enhanced ability to repair DSBs by HR. Other than HR, repair of the ICL-lesions requires additional DNA repair mechanisms, whose action might also be implemented in therapy-resistant cells. This chapter describes GFP assays to measure (a) HR proficiency following formation of a DSB by the endonuclease I-SceI, and (b) HR repair induced by site-specific ICL formation involving psoralen. These experimental approaches can be used to determine the proficiency of TGCT cell lines in DSB repair by HR in comparison to HR repair of ICLs, providing tools to better characterize their recombination profile. Protocols of these assays have been adapted for use in Embryonal Carcinoma (EC) TGCT cell lines. Assays only require transient introduction of plasmids within cells, affording the advantage of testing multiple cell lines in a relatively short time.
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http://dx.doi.org/10.1007/978-1-0716-0860-9_9DOI Listing
March 2021

Interhomolog Homologous Recombination in Mouse Embryonic Stem Cells.

Methods Mol Biol 2021 ;2153:127-143

Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Homologous recombination is a critical mechanism for the repair of DNA double-strand breaks (DSBs). It occurs predominantly between identical sister chromatids and at lower frequency can also occur between homologs. Interhomolog homologous recombination (IH-HR) has the potential lead to substantial loss of genetic information, i.e., loss of heterozygosity (LOH), when it is accompanied by crossing over. In this chapter, we describe a system to study IH-HR induced by a defined DSB in mouse embryonic stem cells derived from F1 hybrid mice. This system is based on the placement of mutant selectable marker genes, one of which contains an I-SceI endonuclease cleavage site, on the two homologs such that repair of the I-SceI-generated DSB from the homolog leads to drug resistance. Loss of heterozygosity arising during IH-HR is analyzed using a PCR-based approach. Finally, we present a strategy to analyze the role of BLM helicase in this system.
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http://dx.doi.org/10.1007/978-1-0716-0644-5_10DOI Listing
March 2021

CRISPR at lightning speeds.

Science 2020 06;368(6496):1180-1181

Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

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http://dx.doi.org/10.1126/science.abc3997DOI Listing
June 2020

Ensuring meiotic DNA break formation in the mouse pseudoautosomal region.

Nature 2020 06 27;582(7812):426-431. Epub 2020 May 27.

Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Sex chromosomes in males of most eutherian mammals share only a small homologous segment, the pseudoautosomal region (PAR), in which the formation of double-strand breaks (DSBs), pairing and crossing over must occur for correct meiotic segregation. How cells ensure that recombination occurs in the PAR is unknown. Here we present a dynamic ultrastructure of the PAR and identify controlling cis- and trans-acting factors that make the PAR the hottest segment for DSB formation in the male mouse genome. Before break formation, multiple DSB-promoting factors hyperaccumulate in the PAR, its chromosome axes elongate and the sister chromatids separate. These processes are linked to heterochromatic mo-2 minisatellite arrays, and require MEI4 and ANKRD31 proteins but not the axis components REC8 or HORMAD1. We propose that the repetitive DNA sequence of the PAR confers unique chromatin and higher-order structures that are crucial for recombination. Chromosome synapsis triggers collapse of the elongated PAR structure and, notably, oocytes can be reprogrammed to exhibit spermatocyte-like levels of DSBs in the PAR simply by delaying or preventing synapsis. Thus, the sexually dimorphic behaviour of the PAR is in part a result of kinetic differences between the sexes in a race between the maturation of the PAR structure, formation of DSBs and completion of pairing and synapsis. Our findings establish a mechanistic paradigm for the recombination of sex chromosomes during meiosis.
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http://dx.doi.org/10.1038/s41586-020-2327-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7337327PMC
June 2020

Mechanistic Insight into Crossing over during Mouse Meiosis.

Mol Cell 2020 06 1;78(6):1252-1263.e3. Epub 2020 May 1.

Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA. Electronic address:

Crossover recombination is critical for meiotic chromosome segregation, but how mammalian crossing over is accomplished is poorly understood. Here, we illuminate how strands exchange during meiotic recombination in male mice by analyzing patterns of heteroduplex DNA in recombinant molecules preserved by the mismatch correction deficiency of Msh2 mutants. Surprisingly, MSH2-dependent recombination suppression was not evident. However, a substantial fraction of crossover products retained heteroduplex DNA, and some provided evidence of MSH2-independent correction. Biased crossover resolution was observed, consistent with asymmetry between DNA ends in earlier intermediates. Many crossover products yielded no heteroduplex DNA, suggesting dismantling by D-loop migration. Unlike the complexity of crossovers in yeast, these simple modifications of the original double-strand break repair model-asymmetry in recombination intermediates and D-loop migration-may be sufficient to explain most meiotic crossing over in mice while also addressing long-standing questions related to Holliday junction resolution.
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http://dx.doi.org/10.1016/j.molcel.2020.04.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7454200PMC
June 2020

Prevention of DNA Replication Stress by CHK1 Leads to Chemoresistance Despite a DNA Repair Defect in Homologous Recombination in Breast Cancer.

Cells 2020 01 17;9(1). Epub 2020 Jan 17.

Laboratory of Radiobiology and Experimental Radiooncology, Center of Oncology, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany.

Chromosomal instability not only has a negative effect on survival in triple-negative breast cancer, but also on the well treatable subgroup of luminal A tumors. This suggests a general mechanism independent of subtypes. Increased chromosomal instability (CIN) in triple-negative breast cancer (TNBC) is attributed to a defect in the DNA repair pathway homologous recombination. Homologous recombination (HR) prevents genomic instability by repair and protection of replication. It is unclear whether genetic alterations actually lead to a repair defect or whether superior signaling pathways are of greater importance. Previous studies focused exclusively on the repair function of HR. Here, we show that the regulation of HR by the intra-S-phase damage response at the replication is of overriding importance. A damage response activated by Ataxia telangiectasia and Rad3 related-checkpoint kinase 1 (ATR-CHK1) can prevent replication stress and leads to resistance formation. CHK1 thus has a preferred role over HR in preventing replication stress in TNBC. The signaling cascade ATR-CHK1 can compensate for a double-strand break repair error and lead to resistance of HR-deficient tumors. Established methods for the identification of HR-deficient tumors for Poly(ADP-Ribose)-Polymerase 1 (PARP1) inhibitor therapies should be extended to include analysis of candidates for intra-S phase damage response.
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http://dx.doi.org/10.3390/cells9010238DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7017274PMC
January 2020

Differential Requirements for the RAD51 Paralogs in Genome Repair and Maintenance in Human Cells.

PLoS Genet 2019 10 4;15(10):e1008355. Epub 2019 Oct 4.

Cancer Research Center of Marseille; CNRS; Inserm; Institut Paoli-Calmettes; Aix-Marseille Université, Marseille, France.

Deficiency in several of the classical human RAD51 paralogs [RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3] is associated with cancer predisposition and Fanconi anemia. To investigate their functions, isogenic disruption mutants for each were generated in non-transformed MCF10A mammary epithelial cells and in transformed U2OS and HEK293 cells. In U2OS and HEK293 cells, viable ablated clones were readily isolated for each RAD51 paralog; in contrast, with the exception of RAD51B, RAD51 paralogs are cell-essential in MCF10A cells. Underlining their importance for genomic stability, mutant cell lines display variable growth defects, impaired sister chromatid recombination, reduced levels of stable RAD51 nuclear foci, and hyper-sensitivity to mitomycin C and olaparib, with the weakest phenotypes observed in RAD51B-deficient cells. Altogether these observations underscore the contributions of RAD51 paralogs in diverse DNA repair processes, and demonstrate essential differences in different cell types. Finally, this study will provide useful reagents to analyze patient-derived mutations and to investigate mechanisms of chemotherapeutic resistance deployed by cancers.
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http://dx.doi.org/10.1371/journal.pgen.1008355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6795472PMC
October 2019

Distinct Requirements of CHD4 during B Cell Development and Antibody Response.

Cell Rep 2019 04;27(5):1472-1486.e5

Immunology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA; Immunology and Microbial Pathogenesis Program, Weill Cornell Graduate School of Medical Sciences, New York, NY, USA; Gerstner Sloan Kettering Graduate School of Biomedical Sciences, New York, NY, USA. Electronic address:

The immunoglobulin heavy chain (Igh) locus features a dynamic chromatin landscape to promote class switch recombination (CSR), yet the mechanisms that regulate this landscape remain poorly understood. CHD4, a component of the chromatin remodeling NuRD complex, directly binds H3K9me3, an epigenetic mark present at the Igh locus during CSR. We find that CHD4 is essential for early B cell development but is dispensable for the homeostatic maintenance of mature, naive B cells. However, loss of CHD4 in mature B cells impairs CSR because of suboptimal targeting of AID to the Igh locus. Additionally, we find that CHD4 represses p53 expression to promote B cell proliferation. This work reveals distinct roles for CHD4 in B cell development and CSR and links the H3K9me3 epigenetic mark with AID recruitment to the Igh locus.
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http://dx.doi.org/10.1016/j.celrep.2019.04.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6527137PMC
April 2019

Under the Influence: Cas9 Ends and DNA Repair Outcomes.

Authors:
Maria Jasin

CRISPR J 2018 04;1:132-134

Developmental Biology Program, Memorial Sloan Kettering Cancer Center , New York, New York.

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http://dx.doi.org/10.1089/crispr.2018.29014.jasDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7477800PMC
April 2018

RAD51D splice variants and cancer-associated mutations reveal XRCC2 interaction to be critical for homologous recombination.

DNA Repair (Amst) 2019 04 23;76:99-107. Epub 2019 Feb 23.

University of Pittsburgh School of Medicine, Department of Microbiology and Molecular Genetics, UPMC Hillman Cancer Center, 5117 Centre Avenue, Pittsburgh, PA, USA. Electronic address:

The proficiency of cancer cells to repair DNA double-strand breaks (DSBs) by homologous recombination (HR) is a key determinant in predicting response to targeted therapies such as PARP inhibitors. The RAD51 paralogs work as multimeric complexes and act downstream of BRCA1 to facilitate HR. Numerous epidemiological studies have linked RAD51 paralog mutations with hereditary cancer predisposition. Despite their substantial links to cancer, RAD51 paralog HR function has remained elusive. Here we identify isoform 1 as the functional isoform of RAD51D, whereas isoform 4 which has a large N-terminal deletion (including the Walker A motif), and isoform 6 which includes an alternate exon in the N-terminus, are non-functional. To determine the importance of this N-terminal region, we investigated the impact of cancer-associated mutations and SNPs in this variable RAD51D N-terminal region using yeast-2-hybrid and yeast-3-hybrid assays to screen for altered protein-protein interactions. We identified two cancer-associated mutations close to or within the Walker A motif (G96C and G107 V, respectively) that independently disrupt RAD51D interaction with XRCC2. We validated our yeast interaction data in human U2OS cells by co-immunoprecipitation and determined the impact of these mutations on HR-proficiency using a sister chromatid recombination reporter assay in a RAD51D knock-out cell line. Our investigation reveals that the interaction of RAD51D with XRCC2 is required for DSB repair. By characterizing the impact of cancer-associated mutations on RAD51D interactions, we aim to develop predictive models for therapeutic sensitivity and resistance in patients who harbor similar mutations in RAD51D.
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http://dx.doi.org/10.1016/j.dnarep.2019.02.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6508892PMC
April 2019

Homology-Directed Repair and the Role of BRCA1, BRCA2, and Related Proteins in Genome Integrity and Cancer.

Annu Rev Cancer Biol 2018 Mar 1;2:313-336. Epub 2017 Dec 1.

Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065.

Germ-line and somatic mutations in genes that promote homology-directed repair (HDR), especially and , are frequently observed in several cancers, in particular, breast and ovary but also prostate and other cancers. HDR is critical for the error-free repair of DNA double-strand breaks and other lesions, and HDR factors also protect stalled replication forks. As a result, loss of BRCA1 or BRCA2 poses significant risks to genome integrity, leading not only to cancer predisposition but also to sensitivity to DNA-damaging agents, affecting therapeutic approaches. Here we review recent advances in our understanding of BRCA1 and BRCA2, including how they genetically interact with other repair factors, how they protect stalled replication forks, how they affect the response to aldehydes, and how loss of their functions links to mutation signatures. Importantly, given the recent advances with poly(ADP-ribose) polymerase inhibitors (PARPi) for the treatment of HDR-deficient tumors, we discuss mechanisms by which BRCA-deficient tumors acquire resistance to PARPi and other agents.
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http://dx.doi.org/10.1146/annurev-cancerbio-030617-050502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6193498PMC
March 2018

Shu complex SWS1-SWSAP1 promotes early steps in mouse meiotic recombination.

Nat Commun 2018 10 10;9(1):3961. Epub 2018 Oct 10.

Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA.

The DNA-damage repair pathway homologous recombination (HR) requires factors that promote the activity of strand-exchange protein RAD51 and its meiosis-specific homolog DMC1. Here we show that the Shu complex SWS1-SWSAP1, a candidate for one such HR regulator, is dispensable for mouse viability but essential for male and female fertility, promoting the assembly of RAD51 and DMC1 on early meiotic HR intermediates. Only a fraction of mutant meiocytes progress to form crossovers, which are crucial for chromosome segregation, demonstrating crossover homeostasis. Remarkably, loss of the DNA damage checkpoint kinase CHK2 rescues fertility in females without rescuing crossover numbers. Concomitant loss of the BRCA2 C terminus aggravates the meiotic defects in Swsap1 mutant spermatocytes, suggesting an overlapping role with the Shu complex during meiotic HR. These results demonstrate an essential role for SWS1-SWSAP1 in meiotic progression and emphasize the complex interplay of factors that ensure recombinase function.
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http://dx.doi.org/10.1038/s41467-018-06384-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6180034PMC
October 2018

Inter-homologue repair in fertilized human eggs?

Nature 2018 08 8;560(7717):E5-E7. Epub 2018 Aug 8.

Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

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http://dx.doi.org/10.1038/s41586-018-0379-5DOI Listing
August 2018

ATR is required to complete meiotic recombination in mice.

Nat Commun 2018 07 5;9(1):2622. Epub 2018 Jul 5.

Genome Integrity and Instability Group, Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, 08193, Spain.

Precise execution of recombination during meiosis is essential for forming chromosomally-balanced gametes. Meiotic recombination initiates with the formation and resection of DNA double-strand breaks (DSBs). Cellular responses to meiotic DSBs are critical for efficient repair and quality control, but molecular features of these remain poorly understood, particularly in mammals. Here we report that the DNA damage response protein kinase ATR is crucial for meiotic recombination and completion of meiotic prophase in mice. Using a hypomorphic Atr mutation and pharmacological inhibition of ATR in vivo and in cultured spermatocytes, we show that ATR, through its effector kinase CHK1, promotes efficient RAD51 and DMC1 assembly at RPA-coated resected DSB sites and establishment of interhomolog connections during meiosis. Furthermore, our findings suggest that ATR promotes local accumulation of recombination markers on unsynapsed axes during meiotic prophase to favor homologous chromosome synapsis. These data reveal that ATR plays multiple roles in mammalian meiotic recombination.
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http://dx.doi.org/10.1038/s41467-018-04851-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6033890PMC
July 2018

Control of meiotic double-strand-break formation by ATM: local and global views.

Cell Cycle 2018 15;17(10):1155-1172. Epub 2018 Jul 15.

a Developmental Biology Program , Memorial Sloan Kettering Cancer Center , New York , NY , USA.

DNA double-strand breaks (DSBs) generated by the SPO11 protein initiate meiotic recombination, an essential process for successful chromosome segregation during gametogenesis. The activity of SPO11 is controlled by multiple factors and regulatory mechanisms, such that the number of DSBs is limited and DSBs form at distinct positions in the genome and at the right time. Loss of this control can affect genome integrity or cause meiotic arrest by mechanisms that are not fully understood. Here we focus on the DSB-responsive kinase ATM and its functions in regulating meiotic DSB numbers and distribution. We review the recently discovered roles of ATM in this context, discuss their evolutionary conservation, and examine future research perspectives.
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http://dx.doi.org/10.1080/15384101.2018.1464847DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6110601PMC
November 2019

Induction of Chromosomal Translocations with CRISPR-Cas9 and Other Nucleases: Understanding the Repair Mechanisms That Give Rise to Translocations.

Adv Exp Med Biol 2018 ;1044:15-25

Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Chromosomal translocations are associated with several tumor types, including hematopoietic malignancies, sarcomas, and solid tumors of epithelial origin, due to their activation of a proto-oncogene or generation of a novel fusion protein with oncogenic potential. In many cases, the availability of suitable human models has been lacking because of the difficulty in recapitulating precise expression of the fusion protein or other reasons. Further, understanding how translocations form mechanistically has been a goal, as it may suggest ways to prevent their occurrence. Chromosomal translocations arise when DNA ends from double-strand breaks (DSBs) on two heterologous chromosomes are improperly joined. This review provides a summary of DSB repair mechanisms and their contribution to translocation formation, the various programmable nuclease platforms that have been used to generate translocations, and the successes that have been achieved in this area.
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http://dx.doi.org/10.1007/978-981-13-0593-1_2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6333474PMC
November 2018

Homologous Recombination and Replication Fork Protection: BRCA2 and More!

Cold Spring Harb Symp Quant Biol 2017 23;82:329-338. Epub 2018 Apr 23.

Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York 10065.

BRCA2 is a breast and ovarian tumor suppressor that guards against genome instability, a hallmark of cancer. Significant progress has been made in improving our understanding of BRCA2 function from biochemical, cellular, and mouse studies. The knowledge gained has been actively exploited to develop therapeutic strategies, including PARP inhibition, which has shown promising clinical outcomes. Recently, tremendous excitement has been generated by the findings of the roles of BRCA2 and other proteins in suppressing replication stress through homologous recombination and in the protection of stalled replication forks. Processes such as mitotic DNA synthesis and fork reversal have taken center stage in these studies. Here, we discuss our recent findings in the context of these advances.
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http://dx.doi.org/10.1101/sqb.2017.82.035006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6333483PMC
April 2018

Inhibition of colorectal cancer genomic copy number alterations and chromosomal fragile site tumor suppressor FHIT and WWOX deletions by DNA mismatch repair.

Oncotarget 2017 Sep 10;8(42):71574-71586. Epub 2017 May 10.

Departments of Medicine and Genetic Medicine, Weill Cornell Medicine, 10021, NY, USA.

Homologous recombination (HR) enables precise DNA repair after DNA double strand breaks (DSBs) using identical sequence templates, whereas homeologous recombination (HeR) uses only partially homologous sequences. Homeologous recombination introduces mutations through gene conversion and genomic deletions through single-strand annealing (SSA). DNA mismatch repair (MMR) inhibits HeR, but the roles of mammalian MMR MutL homologues (MLH1, PMS2 and MLH3) proteins in HeR suppression are poorly characterized. Here, we demonstrate that mouse embryonic fibroblasts (MEFs) carrying , , and mutations have higher HeR rates, by using 7,863 uniquely mapping paired direct repeat sequences (DRs) in the mouse genome as endogenous gene conversion and SSA reporters. Additionally, when DSBs are induced by gamma-radiation, , and mutant MEFs have higher DR copy number alterations (CNAs), including DR CNA hotspots previously identified in mouse MMR-deficient colorectal cancer (dMMR CRC). Analysis of The Cancer Genome Atlas CRC data revealed that dMMR CRCs have higher genome-wide DR HeR rates than MMR proficient CRCs, and that dMMR CRCs have deletion hotspots in tumor suppressors FHIT/WWOX at chromosomal fragile sites and (which have elevated DSB rates) flanked by paired homologous DRs and inverted repeats (IR). Overall, these data provide novel insights into the MMR-dependent HeR inhibition mechanism and its role in tumor suppression.
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http://dx.doi.org/10.18632/oncotarget.17776DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5641073PMC
September 2017

BRCA2 suppresses replication stress-induced mitotic and G1 abnormalities through homologous recombination.

Nat Commun 2017 09 13;8(1):525. Epub 2017 Sep 13.

Developmental Biology Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY, 10065, USA.

Mutations in the tumor suppressor BRCA2 predominantly predispose to breast cancer. Paradoxically, while loss of BRCA2 promotes tumor formation, it also causes cell lethality, although how lethality is triggered is unclear. Here, we generate BRCA2 conditional non-transformed human mammary epithelial cell lines using CRISPR-Cas9. Cells are inviable upon BRCA2 loss, which leads to replication stress associated with under replication, causing mitotic abnormalities, 53BP1 nuclear body formation in the ensuing G1 phase, and G1 arrest. Unexpected from other systems, the role of BRCA2 in homologous recombination, but not in stalled replication fork protection, is primarily associated with supporting human mammary epithelial cell viability, and, moreover, preventing replication stress, a hallmark of pre-cancerous lesions. Thus, we uncover a DNA under replication-53BP1 nuclear body formation-G1 arrest axis as an unanticipated outcome of homologous recombination deficiency, which triggers cell lethality and, we propose, serves as a barrier that must be overcome for tumor formation.BRCA2 mutations promote tumour formation while also paradoxically causing cell lethality. Here the authors generate conditional BRCA2 loss in a non-transformed human mammary cell line and see increased replication stress due to under-replication of DNA.
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http://dx.doi.org/10.1038/s41467-017-00634-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5597640PMC
September 2017

Genomic and chromatin features shaping meiotic double-strand break formation and repair in mice.

Cell Cycle 2017 Oct 18;16(20):1870-1884. Epub 2017 Aug 18.

a Molecular Biology Program, Memorial Sloan Kettering Cancer Center , New York , NY , USA.

The SPO11-generated DNA double-strand breaks (DSBs) that initiate meiotic recombination occur non-randomly across genomes, but mechanisms shaping their distribution and repair remain incompletely understood. Here, we expand on recent studies of nucleotide-resolution DSB maps in mouse spermatocytes. We find that trimethylation of histone H3 lysine 36 around DSB hotspots is highly correlated, both spatially and quantitatively, with trimethylation of H3 lysine 4, consistent with coordinated formation and action of both PRDM9-dependent histone modifications. In contrast, the DSB-responsive kinase ATM contributes independently of PRDM9 to controlling hotspot activity, and combined action of ATM and PRDM9 can explain nearly two-thirds of the variation in DSB frequency between hotspots. DSBs were modestly underrepresented in most repetitive sequences such as segmental duplications and transposons. Nonetheless, numerous DSBs form within repetitive sequences in each meiosis and some classes of repeats are preferentially targeted. Implications of these findings are discussed for evolution of PRDM9 and its role in hybrid strain sterility in mice. Finally, we document the relationship between mouse strain-specific DNA sequence variants within PRDM9 recognition motifs and attendant differences in recombination outcomes. Our results provide further insights into the complex web of factors that influence meiotic recombination patterns.
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http://dx.doi.org/10.1080/15384101.2017.1361065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5638367PMC
October 2017

EXO1 suppresses double-strand break induced homologous recombination between diverged sequences in mammalian cells.

DNA Repair (Amst) 2017 09 10;57:98-106. Epub 2017 Jul 10.

Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY, 10065, USA; Department of Human Science, Georgetown University Medical Center, 3700 Reservoir Rd. NW, Washington, D.C., 20057, USA. Electronic address:

DNA double-strand breaks (DSBs) can be repaired through several mechanisms, including homologous recombination (HR). While HR between identical sequences is robust in mammalian cells, HR between diverged sequences is suppressed by DNA mismatch-repair (MMR) components such as MSH2. Exonuclease I (EXO1) interacts with the MMR machinery and has been proposed to act downstream of the mismatch recognition proteins in mismatch correction. EXO1 has also been shown to participate in extensive DSB end resection, an initial step in the HR pathway. To assess the contribution of EXO1 to HR in mammalian cells, DSB-inducible reporters were introduced into Exo1 mouse embryonic stem cells, including a novel GFP reporter containing several silent polymorphisms to monitor HR between diverged sequences. Compared to HR between identical sequences which was not clearly affected, HR between diverged sequences was substantially increased in Exo1 cells although to a lesser extent than seen in Msh2 cells. Thus, like canonical MMR proteins, EXO1 can restrain aberrant HR events between diverged sequence elements in the genome.
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http://dx.doi.org/10.1016/j.dnarep.2017.07.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5584059PMC
September 2017

ATM loss leads to synthetic lethality in BRCA1 BRCT mutant mice associated with exacerbated defects in homology-directed repair.

Proc Natl Acad Sci U S A 2017 07 28;114(29):7665-7670. Epub 2017 Jun 28.

Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065;

BRCA1 is essential for homology-directed repair (HDR) of DNA double-strand breaks in part through antagonism of the nonhomologous end-joining factor 53BP1. The ATM kinase is involved in various aspects of DNA damage signaling and repair, but how ATM participates in HDR and genetically interacts with BRCA1 in this process is unclear. To investigate this question, we used the mouse model carrying a mutation in the BRCA1 C-terminal domain of BRCA1. Whereas ATM loss leads to a mild HDR defect in adult somatic cells, we find that ATM inhibition leads to severely reduced HDR in cells. Consistent with a critical role for ATM in HDR in this background, loss of ATM leads to synthetic lethality of mice. Whereas both ATM and BRCA1 promote end resection, which can be regulated by 53BP1, deletion does not rescue the HDR defects of mutant cells, in contrast to mutant cells. These results demonstrate that ATM has a role in HDR independent of the BRCA1-53BP1 antagonism and that its HDR function can become critical in certain contexts.
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http://dx.doi.org/10.1073/pnas.1706392114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5530697PMC
July 2017

p53 and TAp63 participate in the recombination-dependent pachytene arrest in mouse spermatocytes.

PLoS Genet 2017 Jun 15;13(6):e1006845. Epub 2017 Jun 15.

Genome Integrity and Instability Group, Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Barcelona, Spain.

To protect germ cells from genomic instability, surveillance mechanisms ensure meiosis occurs properly. In mammals, spermatocytes that display recombination defects experience a so-called recombination-dependent arrest at the pachytene stage, which relies on the MRE11 complex-ATM-CHK2 pathway responding to unrepaired DNA double-strand breaks (DSBs). Here, we asked if p53 family members-targets of ATM and CHK2-participate in this arrest. We bred double-mutant mice combining a mutation of a member of the p53 family (p53, TAp63, or p73) with a Trip13 mutation. Trip13 deficiency triggers a recombination-dependent response that arrests spermatocytes in pachynema before they have incorporated the testis-specific histone variant H1t into their chromatin. We find that deficiency for either p53 or TAp63, but not p73, allowed spermatocytes to progress further into meiotic prophase despite the presence of numerous unrepaired DSBs. Even so, the double mutant spermatocytes apoptosed at late pachynema because of sex body deficiency; thus p53 and TAp63 are dispensable for arrest caused by sex body defects. These data affirm that recombination-dependent and sex body-deficient arrests occur via genetically separable mechanisms.
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http://dx.doi.org/10.1371/journal.pgen.1006845DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5491309PMC
June 2017

Secondary Somatic Mutations Restoring and Associated with Acquired Resistance to the PARP Inhibitor Rucaparib in High-Grade Ovarian Carcinoma.

Cancer Discov 2017 09 6;7(9):984-998. Epub 2017 Jun 6.

Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia.

High-grade epithelial ovarian carcinomas containing mutated or () homologous recombination (HR) genes are sensitive to platinum-based chemotherapy and PARP inhibitors (PARPi), while restoration of HR function due to secondary mutations in has been recognized as an important resistance mechanism. We sequenced core HR pathway genes in 12 pairs of pretreatment and postprogression tumor biopsy samples collected from patients in ARIEL2 Part 1, a phase II study of the PARPi rucaparib as treatment for platinum-sensitive, relapsed ovarian carcinoma. In 6 of 12 pretreatment biopsies, a truncation mutation in , or was identified. In five of six paired postprogression biopsies, one or more secondary mutations restored the open reading frame. Four distinct secondary mutations and spatial heterogeneity were observed for complementation assays and a patient-derived xenograft, as well as predictive molecular modeling, confirmed that resistance to rucaparib was associated with secondary mutations. Analyses of primary and secondary mutations in and provide evidence for these primary mutations in conferring PARPi sensitivity and secondary mutations as a mechanism of acquired PARPi resistance. PARPi resistance due to secondary mutations underpins the need for early delivery of PARPi therapy and for combination strategies. .
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http://dx.doi.org/10.1158/2159-8290.CD-17-0419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5612362PMC
September 2017

Generation of chromosomal translocations that lead to conditional fusion protein expression using CRISPR-Cas9 and homology-directed repair.

Methods 2017 05 15;121-122:138-145. Epub 2017 May 15.

Developmental Biology Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA. Electronic address:

Recurrent chromosomal translocations often lead to expression of fusion proteins associated with oncogenic transformation. To study translocations and downstream events, genome editing techniques have been developed to generate chromosomal translocations through non-homologous end joining of DNA double-strand breaks introduced at the two participating endogenous loci. However, the frequencies at which these events occur is usually too low to efficiently clone cells carrying the translocation. This article provides a detailed method using CRISPR-Cas9 technology and homology-directed repair to efficiently isolate cells harboring a chromosomal translocation. For an additional level of control, the resulting fusion protein is conditionally expressed to allow early events in oncogenic transformation to be studied. We focus on the generation of the EWSR1-WT1 fusion using human mesenchymal cells, which is associated with the translocation found in desmoplastic small round cell tumors.
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http://dx.doi.org/10.1016/j.ymeth.2017.05.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5531069PMC
May 2017

CRISPR-Cas9-guided oncogenic chromosomal translocations with conditional fusion protein expression in human mesenchymal cells.

Proc Natl Acad Sci U S A 2017 04 21;114(14):3696-3701. Epub 2017 Mar 21.

Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065;

Gene editing techniques have been extensively used to attempt to model recurrent genomic rearrangements found in tumor cells. These methods involve the induction of double-strand breaks at endogenous loci followed by the identification of breakpoint junctions within a population, which typically arise by nonhomologous end joining. The low frequency of these events, however, has hindered the cloning of cells with the desired rearrangement before oncogenic transformation. Here we present a strategy combining CRISPR-Cas9 technology and homology-directed repair to allow for the selection of human mesenchymal stem cells harboring the oncogenic translocation found in the aggressive desmoplastic small round cell tumor. The expression of the fusion transcript is under the control of the endogenous promoter and, importantly, can be conditionally expressed using Cre recombinase. This method is easily adapted to generate any cancer-relevant rearrangement.
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http://dx.doi.org/10.1073/pnas.1700622114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389291PMC
April 2017