Homologous Recombination Repair Of DNA Double-strand Breaks Research Articles

ZMYM2 restricts 53BP1 at DNA double-strand breaks to favor BRCA1 loading and homologous recombination.

An inability to repair DNA double-strand breaks (DSBs) threatens genome integrity and can contribute to human diseases, including cancer. Mammalian cells repair DSBs mainly through homologous recombination (HR) and nonhomologous end-joining (NHEJ). The choice between these pathways is regulated by the interplay between 53BP1 and BRCA1, whereby BRCA1 excludes 53BP1 to promote HR and 53BP1 limits BRCA1 to facilitate NHEJ. Here, we identify the zinc-finger proteins (ZnF), ZMYM2 and ZMYM3, as antagonizers of 53BP1 recruitment that facilitate HR protein recruitment and function at DNA breaks. Mechanistically, we show that ZMYM2 recruitment to DSBs and suppression of break-associated 53BP1 requires the SUMO E3 ligase PIAS4, as well as SUMO binding by ZMYM2. Cells deficient for ZMYM2/3 display genome instability, PARP inhibitor and ionizing radiation sensitivity and reduced HR repair. Importantly, depletion of 53BP1 in ZMYM2/3-deficient cells rescues BRCA1 recruitment to and HR repair of DSBs, suggesting that ZMYM2 and ZMYM3 primarily function to restrict 53BP1 engagement at breaks to favor BRCA1 loading that functions to channel breaks to HR repair. Identification of DNA repair functions for these poorly characterized ZnF proteins may shed light on their unknown contributions to human diseases, where they have been reported to be highly dysregulated, including in several cancers.

NRF2 preserves genomic integrity by facilitating ATR activation and G2 cell cycle arrest.

Nuclear factor erythroid 2-related factor 2 (NRF2) is a well-characterized transcription factor that protects cells against oxidative and electrophilic stresses. Emerging evidence has suggested that NRF2 protects cells against DNA damage by mechanisms other than antioxidation, yet the mechanism remains poorly understood. Here, we demonstrate that knockout of NRF2 in cells results in hypersensitivity to ionizing radiation (IR) in the presence or absence of reactive oxygen species (ROS). Under ROS scavenging conditions, induction of DNA double-strand breaks (DSBs) increases the NRF2 protein level and recruits NRF2 to DNA damage sites where it interacts with ATR, resulting in activation of the ATR–CHK1–CDC2 signaling pathway. In turn, this leads to G2 cell cycle arrest and the promotion of homologous recombination repair of DSBs, thereby preserving genome stability. The inhibition of NRF2 by brusatol increased the radiosensitivity of tumor cells in xenografts by perturbing ATR and CHK1 activation. Collectively, our results reveal a novel function of NRF2 as an ATR activator in the regulation of the cellular response to DSBs. This shift in perspective should help furnish a more complete understanding of the function of NRF2 and the DNA damage response.

Identifying and Overcoming Mechanisms of PARP Inhibitor Resistance in Homologous Recombination Repair-Deficient and Repair-Proficient High Grade Serous Ovarian Cancer Cells.

High grade serous ovarian cancer (HGSOC) is a major cause of female cancer mortality. The approval of poly (ADP-ribose) polymerase (PARP) inhibitors for clinical use has greatly improved treatment options for patients with homologous recombination repair (HRR)-deficient HGSOC, although the development of PARP inhibitor resistance in some patients is revealing limitations to outcome. A proportion of patients with HRR-proficient cancers also benefit from PARP inhibitor therapy. Our aim is to compare mechanisms of resistance to the PARP inhibitor olaparib in these two main molecular categories of HGSOC and investigate a way to overcome resistance that we considered particularly suited to a cancer like HGSOC, where there is a very high incidence of TP53 gene mutation, making HGSOC cells heavily reliant on the G2 checkpoint for repair of DNA damage and survival. We identified alterations in multiple factors involved in resistance to PARP inhibition in both HRR-proficient and -deficient cancers. The most frequent change was a major reduction in levels of poly (ADP-ribose) glycohydrolase (PARG), which would be expected to preserve a residual PARP1-initiated DNA damage response to DNA single-strand breaks. Other changes seen would be expected to boost levels of HRR of DNA double-strand breaks. Growth of all olaparib-resistant clones isolated could be controlled by WEE1 kinase inhibitor AZD1775, which inactivates the G2 checkpoint. Our work suggests that use of the WEE1 kinase inhibitor could be a realistic therapeutic option for patients that develop resistance to olaparib.

Checkpoint Kinase 1 (CHK1) Inhibition Enhances the Sensitivity of Triple-Negative Breast Cancer Cells to Proton Irradiation via Rad51 Downregulation.

Due to a superior dose conformity to the target, proton beam therapy (PBT) continues to rise in popularity. Recently, considerable efforts have been directed toward discovering treatment options for use in combination with PBT. This study aimed to investigate the targeting of checkpoint kinase 1 (CHK1), a critical player regulating the G2/M checkpoint, as a promising strategy to potentiate PBT in human triple-negative breast cancer (TNBC) cells. Protons induced cell-cycle arrest at the G2/M checkpoint more readily in response to increased CHK1 activation than X-rays. A clonogenic survival assay revealed that CHK1 inhibition using PF-477736 or small interfering RNA (siRNA) enhanced the sensitivity toward protons to a greater extent than toward X-rays. Western blotting demonstrated that PF-477736 treatment in the background of proton irradiation increased the pro-apoptotic signaling, which was further supported by flow cytometry using annexin V. Immunofluorescence revealed that proton-induced DNA double-strand breaks (DSBs) were further enhanced by PF-477736, which was linked to the downregulation of Rad51, essential for the homologous recombination repair of DSBs. Direct inactivation of Rad51 resulted in enhanced proton sensitization. Collectively, these data suggest that targeting CHK1 may be a promising approach for improving PBT efficacy in the treatment of TNBC.

SETD2 is required for DNA double-strand break repair and activation of the p53-mediated checkpoint.

Histone modifications establish the chromatin states that coordinate the DNA damage response. In this study, we show that SETD2, the enzyme that trimethylates histone H3 lysine 36 (H3K36me3), is required for ATM activation upon DNA double-strand breaks (DSBs). Moreover, we find that SETD2 is necessary for homologous recombination repair of DSBs by promoting the formation of RAD51 presynaptic filaments. In agreement, SETD2-mutant clear cell renal cell carcinoma (ccRCC) cells displayed impaired DNA damage signaling. However, despite the persistence of DNA lesions, SETD2-deficient cells failed to activate p53, a master guardian of the genome rarely mutated in ccRCC and showed decreased cell survival after DNA damage. We propose that this novel SETD2-dependent role provides a chromatin bookmarking instrument that facilitates signaling and repair of DSBs. In ccRCC, loss of SETD2 may afford an alternative mechanism for the inactivation of the p53-mediated checkpoint without the need for additional genetic mutations in TP53.DOI: http://dx.doi.org/10.7554/eLife.02482.001.

Targeting Aberrant DNA Double-Strand Break Repair in Triple-Negative Breast Cancer with Alpha-Particle Emitter Radiolabeled Anti-EGFR Antibody

The higher potential efficacy of alpha-particle radiopharmaceutical therapy lies in the 3- to 8-fold greater relative biological effectiveness (RBE) of alpha particles relative to photon or beta-particle radiation. This greater RBE, however, also applies to normal tissue, thereby reducing the potential advantage of high RBE. As alpha particles typically cause DNA double-strand breaks (DSB), targeting tumors that are defective in DSB repair effectively increases the RBE, yielding a secondary, RBE-based differentiation between tumor and normal tissue that is complementary to conventional, receptor-mediated tumor targeting. In some triple-negative breast cancers (TNBC; ER(-)/PR(-)/HER-2(-)), germline mutation in BRCA-1, a key gene in homologous recombination DSB repair, predisposes patients to early onset of breast cancer. These patients have few treatment options once the cancer has metastasized. In this study, we investigated the efficacy of alpha-particle emitter, (213)Bi-labeled anti-EGF receptor antibody, cetuximab, in BRCA-1-defective TNBC. (213)Bi-cetuximab was found to be significantly more effective in the BRCA-1-mutated TNBC cell line HCC1937 than BRCA-1-competent TNBC cell MDA-MB-231. siRNA knockdown of BRCA-1 or DNA-dependent protein kinase, catalytic subunit (DNA-PKcs), a key gene in non-homologous end-joining DSB repair pathway, also sensitized TNBC cells to (213)Bi-cetuximab. Furthermore, the small-molecule inhibitor of DNA-PKcs, NU7441, sensitized BRCA-1-competent TNBC cells to alpha-particle radiation. Immunofluorescent staining of γ-H2AX foci and comet assay confirmed that enhanced RBE is caused by impaired DSB repair. These data offer a novel strategy for enhancing conventional receptor-mediated targeting with an additional, potentially synergistic radiobiological targeting that could be applied to TNBC.

Abstract 2743: Triapine sensitizes ovarian cancer cells to platinum drugs and PARP inhibitors

Abstract Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone) is a ribonucleotide reductase (RNR) inhibitor that is 1000-times more potent than clinically-used hydroxyurea. Consisting primarily of R2 and R1 subunits during the S phase of the cell cycle, RNR catalyzes the rate-limiting step in the production of deoxyribonucleotides (dNTPs) required for DNA synthesis. Triapine chelates iron to form the triapine-Fe complex that quenches the tyrosyl radical at the R2 subunit and leads to enzymatic inactivation. In recent phase I and II trials, treatment of locally advanced cervical cancer with triapine in combination with cisplatin and radiation was well tolerated and produced high durable complete clinical responses in patients. This promising combination therapy may be attributable to the inhibitory effects of triapine on the repair of cisplatin- and radiation-induced DNA damage. However, the exact DNA repair mechanisms modulated by triapine remain poorly understood. In this study, we demonstrate that treatment of ovarian cancer cells with triapine prompts rapid and prolonged depletion of dATP/dNTP pools, which in turn leads to formation of DNA double strand breaks (DSBs) and inhibition of DNA synthesis in S and G2/M phases of the cell cycle. Furthermore, we show that treatment with triapine results in enhanced sensitization of BRCA-proficient (wild-type) ovarian cancer cells to platinum drugs (cisplatin and carboplatin) and the PARP inhibitor olaparib. Both platinum drugs and PARP inhibitors indirectly cause DSBs which require BRCA1- and BRCA2-dependent homologous recombination (HR) to repair. Collectively, these results suggest that triapine acts to abrogate replicative DNA synthesis and post-replication repair, thereby deterring HR repair of DSBs caused by platinum drugs and other DNA damaging modalities. While 70% of ovarian cancer patients initially respond to platinum-based chemotherapy, the majority of these patients will develop recurrent cancer resistant to the platinum drugs. Emergence of platinum-resistant cancer populations may be associated with increasing proficiency of cancer cells to employ HR to repair DNA damage. Given that, our findings provide the rationale for the use of triapine to enhance the efficacy of platinum-based chemotherapy and PARP inhibitors, and potentially eradicate platinum-resistant ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2743. doi:1538-7445.AM2012-2743

A Genome-Scale DNA Repair RNAi Screen Identifies SPG48 as a Novel Gene Associated with Hereditary Spastic Paraplegia

Author SummaryAll cells in our bodies have to cope with numerous lesions to their DNA. Cells use a battery of genes to repair DNA and maintain genome integrity. Given the importance of an intact genome, it is not surprising that genes with roles in DNA repair are mutated in many human diseases. Here, we present the results of a genome-scale DNA repair screen in human cells and discover 61 genes that have a potential role in this process. We studied in detail a previously uncharacterized gene (KIAA0415/SPG48) and demonstrated its importance for efficient DNA double strand break repair. Further analyses revealed mutations in the SPG48 gene in some patients with hereditary spastic paraplegia (HSP). We showed that SPG48 physically interacts with other HSP proteins and that patient cells are sensitive to DNA damaging drugs. Our data suggest a link between HSP and DNA repair and we propose that HSP patients should be screened for KIAA0415/SPG48 mutations in the future.

Distinct RAD51 Associations with RAD52 and BCCIP in Response to DNA Damage and Replication Stress

RAD51 has critical roles in homologous recombination (HR) repair of DNA double-strand breaks (DSB) and restarting stalled or collapsed replication forks. In yeast, Rad51 function is facilitated by Rad52 and other "mediators." Mammalian cells express RAD52, but BRCA2 may have supplanted RAD52 in mediating RAD51 loading onto ssDNA. BCCIP interacts with BRCA2, and both proteins are important for RAD51 focus formation after ionizing radiation and HR repair of DSBs. Nonetheless, mammalian RAD52 shares biochemical activities with yeast Rad52, including RAD51 binding and single-strand annealing, suggesting a conserved role in HR. Because RAD52 and RAD51 associate, and RAD51 and BCCIP associate, we investigated the colocalization of RAD51 with BCCIP and RAD52 in human cells. We found that RAD51 colocalizes with BCCIP early after ionizing radiation, with RAD52 later, and there was little colocalization of BCCIP and RAD52. RAD52 foci are induced to a greater extent by hydroxyurea, which stalls replication forks, than by ionizing radiation. Using fluorescence recovery after photo bleaching, we show that RAD52 mobility is reduced to a greater extent by hydroxyurea than ionizing radiation. However, BCCIP showed no changes in mobility after hydroxyurea or ionizing radiation. We propose that BCCIP-dependent repair of DSBs by HR is an early RAD51 response to ionizing radiation-induced DNA damage, and that RAD52-dependent HR occurs later to restart a subset of blocked or collapsed replication forks. RAD52 and BRCA2 seem to act in parallel pathways, suggesting that targeting RAD52 in BRCA2-deficient tumors may be effective in treating these tumors.

141 INDICATIONS FOR DNA DOUBLE-STRAND BREAK REPAIR IN EARLY BOVINE EMBRYOS

DNA double-strand breaks (DSBs) are considered the most severe type of DNA lesions, because such lesions, if unrepaired, lead to a loss of genome integrity. Soon after induction of DSBs, chromatin surrounding the damage is modified by phosphorylation of the histone variant H2AX, generating so-called γH2AX, which is a hallmark of DSBs (Takahashi et al. 2005 Cancer Lett. 229, 171–179). γH2AX appears to be a signal for the recruitment of proteins constituting the DNA repair machinery. Depending on the type of damage and the cell cycle stage of the affected cell, DSBs are repaired either by nonhomologous end joining or by homologous recombination using the sister chromatid DNA as template (Hoeijmakers 2001 Nature 411, 366–374). We used immunofluorescence to analyze chromatin composition during bovine development and found γH2AX foci in both male and female pronuclei of IVF embryos. The number and size of foci varied considerably between embryos and between the male and female pronuclei. To test whether the observed γH2AX foci represented sites of active DNA repair, we co-stained IVF zygotes for γH2AX and 3 different proteins involved in homologous recombination repair of DSBs: NBS1 (phosphorylated at amino acid serine 343), 53BP1, and Rad51. We found co-localization of γH2AX foci with phosphorylated NBS1 as well as with Rad51 but did not observe the presence of 53BP1 at γH2AX foci in IVF zygotes. Our finding shows the presence of DSBs in IVF zygotes and suggests the capability of homologous recombination repair. The lack of 53BP1, a component of homologous recombination repair, which usually co-localizes with γH2AX foci at exogenously induced DSBs (Schultz et al. 2000 J. Cell. Biol. 151, 1381–1390) poses the possibility that the mechanism present in early embryos differs substantially from that involved in DNA repair of DSBs in somatic cells.