Browsing by Subject "Homologous recombination"
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Item Defective Resection at DNA Double-Strand Breaks Leads to De Novo Telomere Formation and Enhances Gene Targeting(Public Library of Science, 2010-05-13) Chung, Woo-Hyun; Zhu, Zhu; Papusha, Alma; Malkova, Anna; Ira, Grzegorz; Biology, School of ScienceThe formation of single-stranded DNA (ssDNA) at double-strand break (DSB) ends is essential in repair by homologous recombination and is mediated by DNA helicases and nucleases. Here we estimated the length of ssDNA generated during DSB repair and analyzed the consequences of elimination of processive resection pathways mediated by Sgs1 helicase and Exo1 nuclease on DSB repair fidelity. In wild-type cells during allelic gene conversion, an average of 2–4 kb of ssDNA accumulates at each side of the break. Longer ssDNA is formed during ectopic recombination or break-induced replication (BIR), reflecting much slower repair kinetics. This relatively extensive resection may help determine sequences involved in homology search and prevent recombination within short DNA repeats next to the break. In sgs1Δ exo1Δ mutants that form only very short ssDNA, allelic gene conversion decreases 5-fold and DSBs are repaired by BIR or de novo telomere formation resulting in loss of heterozygosity. The absence of the telomerase inhibitor, PIF1, increases de novo telomere pathway usage to about 50%. Accumulation of Cdc13, a protein recruiting telomerase, at the break site increases in sgs1Δ exo1Δ, and the requirement of the Ku complex for new telomere formation is partially bypassed. In contrast to this decreased and alternative DSB repair, the efficiency and accuracy of gene targeting increases dramatically in sgs1Δ exo1Δ cells, suggesting that transformed DNA is very stable in these mutants. Altogether these data establish a new role for processive resection in the fidelity of DSB repair.Item Drugging the Cancers Addicted to DNA Repair(Oxford University Press, 2017-11-01) Nickoloff, Jac A.; Jones, Dennie; Lee, Suk-Hee; Williamson, Elizabeth A.; Hromas, Robert; Department of Biochemistry and Molecular Biology, School of MedicineDefects in DNA repair can result in oncogenic genomic instability. Cancers occurring from DNA repair defects were once thought to be limited to rare inherited mutations (such as BRCA1 or 2). It now appears that a clinically significant fraction of cancers have acquired DNA repair defects. DNA repair pathways operate in related networks, and cancers arising from loss of one DNA repair component typically become addicted to other repair pathways to survive and proliferate. Drug inhibition of the rescue repair pathway prevents the repair-deficient cancer cell from replicating, causing apoptosis (termed synthetic lethality). However, the selective pressure of inhibiting the rescue repair pathway can generate further mutations that confer resistance to the synthetic lethal drugs. Many such drugs currently in clinical use inhibit PARP1, a repair component to which cancers arising from inherited BRCA1 or 2 mutations become addicted. It is now clear that drugs inducing synthetic lethality may also be therapeutic in cancers with acquired DNA repair defects, which would markedly broaden their applicability beyond treatment of cancers with inherited DNA repair defects. Here we review how each DNA repair pathway can be attacked therapeutically and evaluate DNA repair components as potential drug targets to induce synthetic lethality. Clinical use of drugs targeting DNA repair will markedly increase when functional and genetic loss of repair components are consistently identified. In addition, future therapies will exploit artificial synthetic lethality, where complementary DNA repair pathways are targeted simultaneously in cancers without DNA repair defects.Item Endonuclease EEPD1 Is a Gatekeeper for Repair of Stressed Replication Forks(American Society for Biochemistry and Molecular Biology, 2017-02-17) Kim, Hyun-Suk; Nickoloff, Jac A.; Wu, Yuehan; Williamson, Elizabeth A.; Sidhu, Gurjit Singh; Reinart, Brian L.; Jaiswal, Aruna S.; Srinivasan, Gayathri; Patel, Bhavita; Kong, Kimi; Burma, Sandeep; Lee, Suk-Hee; Hromas, Robert A.; Department of Biochemistry & Molecular Biology, IU School of MedicineReplication is not as continuous as once thought, with DNA damage frequently stalling replication forks. Aberrant repair of stressed replication forks can result in cell death or genome instability and resulting transformation to malignancy. Stressed replication forks are most commonly repaired via homologous recombination (HR), which begins with 5' end resection, mediated by exonuclease complexes, one of which contains Exo1. However, Exo1 requires free 5'-DNA ends upon which to act, and these are not commonly present in non-reversed stalled replication forks. To generate a free 5' end, stalled replication forks must therefore be cleaved. Although several candidate endonucleases have been implicated in cleavage of stalled replication forks to permit end resection, the identity of such an endonuclease remains elusive. Here we show that the 5'-endonuclease EEPD1 cleaves replication forks at the junction between the lagging parental strand and the unreplicated DNA parental double strands. This cleavage creates the structure that Exo1 requires for 5' end resection and HR initiation. We observed that EEPD1 and Exo1 interact constitutively, and Exo1 repairs stalled replication forks poorly without EEPD1. Thus, EEPD1 performs a gatekeeper function for replication fork repair by mediating the fork cleavage that permits initiation of HR-mediated repair and restart of stressed forks.Item The endonuclease EEPD1 mediates synthetic lethality in RAD52-depleted BRCA1 mutant breast cancer cells(BMC, 2017) Hromas, Robert; Kim, Hyun-Suk; Sidhu, Gurjit; Williamson, Elizabeth; Jaiswal, Aruna; Totterdale, Taylor A.; Nole, Jocelyn; Lee, Suk-Hee; Nickoloff, Jac A.; Kong, Kimi Y.; Biochemistry and Molecular Biology, School of MedicineBackground Proper repair and restart of stressed replication forks requires intact homologous recombination (HR). HR at stressed replication forks can be initiated by the 5′ endonuclease EEPD1, which cleaves the stalled replication fork. Inherited or acquired defects in HR, such as mutations in breast cancer susceptibility protein-1 (BRCA1) or BRCA2, predispose to cancer, including breast and ovarian cancers. In order for these HR-deficient tumor cells to proliferate, they become addicted to a bypass replication fork repair pathway mediated by radiation repair protein 52 (RAD52). Depleting RAD52 can cause synthetic lethality in BRCA1/2 mutant cancers by an unknown molecular mechanism. Methods We hypothesized that cleavage of stressed replication forks by EEPD1 generates a fork repair intermediate that is toxic when HR-deficient cells cannot complete repair with the RAD52 bypass pathway. To test this hypothesis, we applied cell survival assays, immunofluorescence staining, DNA fiber and western blot analyses to look at the correlation between cell survival and genome integrity in control, EEPD1, RAD52 and EEPD1/RAD52 co-depletion BRCA1-deficient breast cancer cells. Results Our data show that depletion of EEPD1 suppresses synthetic lethality, genome instability, mitotic catastrophe, and hypersensitivity to stress of replication of RAD52-depleted, BRCA1 mutant breast cancer cells. Without HR and the RAD52-dependent backup pathway, the BRCA1 mutant cancer cells depleted of EEPD1 skew to the alternative non-homologous end-joining DNA repair pathway for survival. Conclusion This study indicates that the mechanism of synthetic lethality in RAD52-depleted BRCA1 mutant cancer cells depends on the endonuclease EEPD1. The data imply that EEPD1 cleavage of stressed replication forks may result in a toxic intermediate when replication fork repair cannot be completed. Electronic supplementary material The online version of this article (doi:10.1186/s13058-017-0912-8) contains supplementary material, which is available to authorized users.Item Inherited DNA-Repair Defects in Colorectal Cancer(Elsevier, 2018-03-01) AlDubayan, Saud H.; Giannakis, Marios; Moore, Nathanael D.; Han, G. Celine; Reardon, Brendan; Hamada, Tsuyoshi; Mu, Xingmeng Jasmine; Nishihara, Reiko; Qian, Zhirong; Liu, Li; Yurgelun, Matthew B.; Syngal, Sapna; Garraway, Levi A.; Ogino, Shuji; Fuchs, Charles S.; Van Allen, Eliezer M.; Medicine, School of MedicineColorectal cancer (CRC) heritability has been estimated to be around 30%. However, mutations in the known CRC-susceptibility genes explain CRC risk in fewer than 10% of affected individuals. Germline mutations in DNA-repair genes (DRGs) have recently been reported in CRC, but their contribution to CRC risk is largely unknown. We evaluated the gene-level germline mutation enrichment of 40 DRGs in 680 unselected CRC individuals and 27,728 ancestry-matched cancer-free adults. Significant findings were then examined in independent cohorts of 1,661 unselected CRC individuals and 1,456 individuals with early-onset CRC. Of the 680 individuals in the discovery set, 31 (4.56%) individuals harbored germline pathogenic mutations in known CRC-susceptibility genes, and another 33 (4.85%) individuals had DRG mutations that have not been previously associated with CRC risk. Germline pathogenic mutations in ATM and PALB2 were enriched in both the discovery (OR = 2.81 and p = 0.035 for ATM and OR = 4.91 and p = 0.024 for PALB2) and validation (OR = 2.97 and adjusted p = 0.0013 for ATM and OR = 3.42 and adjusted p = 0.034 for PALB2) sets. Biallelic loss of ATM was evident in all individuals with matched tumor profiling. CRC individuals also had higher rates of actionable mutations in the HR pathway, which can substantially increase the risk of developing cancers other than CRC. Our analysis provides evidence for ATM and PALB2 as CRC-risk genes, underscoring the importance of the homologous recombination pathway in CRC. In addition, we identified frequent complete homologous recombination deficiency in CRC tumors, representing a unique opportunity to explore targeted therapeutic interventions such as poly-ADP ribose polymerase inhibitor (PARPi).Item Metnase and EEPD1: DNA Repair Functions and Potential Targets in Cancer Therapy(Frontiers Media, 2022-01-28) Nickoloff, Jac A.; Sharma, Neelam; Taylor, Lynn; Allen, Sage J.; Lee, Suk-Hee; Hromas, Robert; Biochemistry and Molecular Biology, School of MedicineCells respond to DNA damage by activating signaling and DNA repair systems, described as the DNA damage response (DDR). Clarifying DDR pathways and their dysregulation in cancer are important for understanding cancer etiology, how cancer cells exploit the DDR to survive endogenous and treatment-related stress, and to identify DDR targets as therapeutic targets. Cancer is often treated with genotoxic chemicals and/or ionizing radiation. These agents are cytotoxic because they induce DNA double-strand breaks (DSBs) directly, or indirectly by inducing replication stress which causes replication fork collapse to DSBs. EEPD1 and Metnase are structure-specific nucleases, and Metnase is also a protein methyl transferase that methylates histone H3 and itself. EEPD1 and Metnase promote repair of frank, two-ended DSBs, and both promote the timely and accurate restart of replication forks that have collapsed to single-ended DSBs. In addition to its roles in HR, Metnase also promotes DSB repair by classical non-homologous recombination, and chromosome decatenation mediated by TopoIIα. Although mutations in Metnase and EEPD1 are not common in cancer, both proteins are frequently overexpressed, which may help tumor cells manage oncogenic stress or confer resistance to therapeutics. Here we focus on Metnase and EEPD1 DNA repair pathways, and discuss opportunities for targeting these pathways to enhance cancer therapy.Item Replication gaps are a key determinant of PARP inhibitor synthetic lethality with BRCA deficiency(Cell Press, 2021) Cong, Ke; Peng, Min; Kousholt, Arne Nedergaard; Lee, Wei Ting C.; Lee, Silviana; Nayak, Sumeet; Krais, John; VanderVere-Carozza, Pamela S.; Pawelczak, Katherine S.; Calvo, Jennifer; Panzarino, Nicholas J.; Turchi, John J.; Johnson, Neil; Jonkers, Jos; Rothenberg, Eli; Cantor, Sharon B.; Medicine, School of MedicineMutations in BRCA1 or BRCA2 (BRCA) is synthetic lethal with poly(ADP-ribose) polymerase inhibitors (PARPi). Lethality is thought to derive from DNA double-stranded breaks (DSBs) necessitating BRCA function in homologous recombination (HR) and/or fork protection (FP). Here, we report instead that toxicity derives from replication gaps. BRCA1- or FANCJ-deficient cells, with common repair defects but distinct PARPi responses, reveal gaps as a distinguishing factor. We further uncouple HR, FP, and fork speed from PARPi response. Instead, gaps characterize BRCA-deficient cells, are diminished upon resistance, restored upon resensitization, and, when exposed, augment PARPi toxicity. Unchallenged BRCA1-deficient cells have elevated poly(ADP-ribose) and chromatin-associated PARP1, but aberrantly low XRCC1 consistent with defects in backup Okazaki fragment processing (OFP). 53BP1 loss resuscitates OFP by restoring XRCC1-LIG3 that suppresses the sensitivity of BRCA1-deficient cells to drugs targeting OFP or generating gaps. We highlight gaps as a determinant of PARPi toxicity changing the paradigm for synthetic lethal interactions.Item A Role for SSRP1 in Recombination-Mediated DNA Damage Response(Wiley, 2009-10) Kumari, Anuradha; Mazina, Olga M.; Shinde, Ujwal; Mazin, Alexander V.; Lu, Hua; Biochemistry and Molecular Biology, School of MedicineA possible role for structure-specific recognition protein 1 (SSRP1) in replication-associated repair processes has previously been suggested based on its interaction with several DNA repair factors and the replication defects observed in SSRP1 mutants. In this study, we investigated the potential role of SSRP1 in association with DNA repair mediated by homologous recombination (HR), one of the pathways involved in repairing replication-associated DNA damage, in mammalian cells. Surprisingly, over-expression of SSRP1 reduced the number of hprt(+) recombinants generated via HR both spontaneously and upon hydroxyurea (HU) treatment, whereas knockdown of SSRP1 resulted in an increase of HR events in response to DNA double-strand break formation. In correlation, we found that the depletion of SSRP1 in HU-treated human cells elevated the number of Rad51 and H2AX foci, while over-expression of the wild-type SSRP1 markedly reduced HU-induced Rad51 foci formation. We also found that SSRP1 physically interacts with a key HR repair protein, Rad54 both in vitro and in vivo. Further, branch migration studies demonstrated that SSRP1 inhibits Rad54-promoted branch migration of Holliday junctions in vitro. Taken together, our data suggest a functional role for SSRP1 in spontaneous and replication-associated DNA damage response by suppressing avoidable HR repair events.