Defect of MLH1 expression sensitized esophageal squamous cell carcinoma cells to Polθ inhibitor.

A Cell Cycle-Dependent Regulatory Circuit Composed of 53BP1-RIF1 and BRCA1-CtIP Controls DNA Repair Pathway Choice

ATM Signaling Facilitates Repair of DNA Double-Strand Breaks Associated with Heterochromatin

Requirement of ATM-Dependent Monoubiquitylation of Histone H2B for Timely Repair of DNA Double-Strand Breaks

Decision letter: SPOP mutation leads to genomic instability in prostate cancer

Up-Regulated WRN and DNA Ligase IIIα Are Involved in Alternative NHEJ Repair Pathway of DNA Double Strand Breaks (DSB) in Chronic Myeloid Leukemia (CML).

The tumor suppressor breast cancer susceptibility protein 1 (BRCA1) protects our cells from genomic instability in part by facilitating the efficient repair of DNA double-strand breaks (DSBs). BRCA1 promotes the error-free repair of DSBs through homologous recombination and is also implicated in the regulation of nonhomologous end joining (NHEJ) repair fidelity. Here, we investigate the role of BRCA1 in NHEJ repair mutagenesis following a DSB. We examined the frequency of microhomology-mediated end joining (MMEJ) and the fidelity of DSB repair relative to BRCA1 protein levels in both control and tumorigenic breast epithelial cells. In addition to altered BRCA1 protein levels, we tested the effects of cellular exposure to mirin, an inhibitor of meiotic recombination enzyme 11 (Mre11) 3'-5'-exonuclease activity. Knockdown or loss of BRCA1 protein resulted in an increased frequency of overall plasmid DNA mutagenesis and MMEJ following a DSB. Inhibition of Mre11-exonuclease activity with mirin significantly decreased the occurrence of MMEJ, but did not considerably affect the overall mutagenic frequency of plasmid DSB repair. The results suggest that BRCA1 protects DNA from mutagenesis during nonhomologous DSB repair in plasmid-based assays. The increased frequency of DSB mutagenesis and MMEJ repair in the absence of BRCA1 suggests a potential mechanism for carcinogenesis.

What a 'Ku'incidence!: parallel discoveries of a new DNA repair factor.

DNA non-homologous end joining (NHEJ) and homologous recombination (HR) function to repair DNA double-strand breaks (DSBs) in G2 phase with HR preferentially repairing heterochromatin-associated DSBs (HC-DSBs). Here, we examine the regulation of repair pathway usage at two-ended DSBs in G2. We identify the speed of DSB repair as a major component influencing repair pathway usage showing that DNA damage and chromatin complexity are factors influencing DSB repair rate and pathway choice. Loss of NHEJ proteins also slows DSB repair allowing increased resection. However, expression of an autophosphorylation-defective DNA-PKcs mutant, which binds DSBs but precludes the completion of NHEJ, dramatically reduces DSB end resection at all DSBs. In contrast, loss of HR does not impair repair by NHEJ although CtIP-dependent end resection precludes NHEJ usage. We propose that NHEJ initially attempts to repair DSBs and, if rapid rejoining does not ensue, then resection occurs promoting repair by HR. Finally, we identify novel roles for ATM in regulating DSB end resection; an indirect role in promoting KAP-1-dependent chromatin relaxation and a direct role in phosphorylating and activating CtIP.

Regulation of ATM in DNA double strand break repair accounts for the radiosensitivity in human cells exposed to high linear energy transfer ionizing radiation

Fisetin overcomes non-targetability of mutated KRAS induced YB-1 signaling in colorectal cancer cells and improves radiosensitivity by blocking repair of radiation-induced DNA double-strand breaks

Development of a novel method to create double-strand break repair fingerprints using next-generation sequencing.

Ataxia telangiectasia mutated (ATM) is a serine–threonine protein kinase and important regulator of the DNA damage response (DDR). One critical ATM target is the structural subunit A (PR65–S401) of protein phosphatase 2A (PP2A), known to regulate diverse cellular processes such as mitosis and cell growth as well as dephosphorylating many proteins during the recovery from the DDR. We generated mouse embryonic fibroblasts expressing PR65-WT, -S401A (cannot be phosphorylated), and -S401D (phospho-mimetic) transgenes. Significantly, S401 mutants exhibited extensive chromosomal aberrations, impaired DNA double-strand break (DSB) repair and underwent increased mitotic catastrophe after radiation. Both S401A and the S401D cells showed impaired DSB repair (nonhomologous end joining and homologous recombination repair) and exhibited delayed DNA damage recovery, which was reflected in reduced radiation survival. Furthermore, S401D cells displayed increased ERK and AKT signaling resulting in enhanced growth rate further underscoring the multiple roles ATM–PP2A signaling plays in regulating prosurvival responses. Time-lapse video and cellular localization experiments showed that PR65 was exported to the cytoplasm after radiation by CRM1, a nuclear export protein, in line with the very rapid pleiotropic effects observed. A putative nuclear export sequence (NES) close to S401 was identified and when mutated resulted in aberrant PR65 shuttling. Our study demonstrates that the phosphorylation of a single, critical PR65 amino acid (S401) by ATM fundamentally controls the DDR, and balances DSB repair quality, cell survival and growth by spatiotemporal PR65 nuclear–cytoplasmic shuttling mediated by the nuclear export receptor CRM1.

Microhomology-mediated end joining (MMEJ), an error-prone pathway for DNA double-strand break (DSB) repair, is implicated in genomic rearrangement and oncogenic transformation; however, its contribution to repair of radiation-induced DSBs has not been characterized. We used recircularization of a linearized plasmid with 3΄-P-blocked termini, mimicking those at X-ray-induced strand breaks, to recapitulate DSB repair via MMEJ or nonhomologous end-joining (NHEJ). Sequence analysis of the circularized plasmids allowed measurement of relative activity of MMEJ versus NHEJ. While we predictably observed NHEJ to be the predominant pathway for DSB repair in our assay, MMEJ was significantly enhanced in preirradiated cells, independent of their radiation-induced arrest in the G2/M phase. MMEJ activation was dependent on XRCC1 phosphorylation by casein kinase 2 (CK2), enhancing XRCC1's interaction with the end resection enzymes MRE11 and CtIP. Both endonuclease and exonuclease activities of MRE11 were required for MMEJ, as has been observed for homology-directed DSB repair (HDR). Furthermore, the XRCC1 co-immunoprecipitate complex (IP) displayed MMEJ activity in vitro, which was significantly elevated after irradiation. Our studies thus suggest that radiation-mediated enhancement of MMEJ in cells surviving radiation therapy may contribute to their radioresistance and could be therapeutically targeted.

ATM and 53BP1 Function in Nonoverlapping Pathways to Suppress T Lineage Lymphomagenesis.

PARP inhibitors (PARPi) have exhibited clinical success in inherited breast cancers with mutations in homologous recombination (HR) DNA double strand break (DSB) repair genes BRCA 1/2 by synthetic lethality. Novel PARPi have been shown to function not only by catalytic inhibition of PARP 1/2 activity, but also by “trapping” of PARP to sites of DNA damage. Trapping of PARP is a response generated by PARPi that is independent of the inhibition of PARP’s catalytic activity, and leads to lethal unrepaired DSBs during replication. Importantly, PARP trapping ability of PARPi correlates with its cytotoxic effect. In this study, we investigated whether PARP trapping has distinct effects on regulation of DNA DSB repair in breast cancer cells. Major DSB repair pathways in mammalian cells include HR and non-homologous end joining (c-NHEJ). Alternative NHEJ (ALT-NHEJ) is a recently discovered and highly error-prone pathway which requires DNA resection like HR, and utilizes PARP1. Our hypothesis is that PARP1 catalytic inhibition vs ‘trapping’ leads to distinct outcomes on DSB repair, highlighting inhibitor efficacy. To test our hypothesis, we utilized MCF10A (non-tumorigenic), MDA-MB-231 (TNBC/BRCA+), and SUM149PT (TNBC/BRCA-) breast cancer cell lines with stably integrated GFP-reporter plasmids designed to assess interchromosomal activities of the specific DSB repair pathways. For our analysis, we used varying concentrations of Talazoparib (MDV3800), which is a potent PARP trapping agent when used in combination with DNA damaging agents such as methyl methanesulfonate (MMS). First, we determined optimal conditions to induce PARP trapping in cells without leading to significant toxicity. As observed by others, combination treatments with of MMS/MDV3800 led to a significant increase in PARP trapping relative to treatments with individual drugs. Studies were also performed with a potent inhibitor of coenzyme NAD+ (Daporinad/FK866) that blocks poly-ADP ribosylation activity in combination of MMS. We observed significant PARP trapping with FK866/MMS combination treatment, indicating that PARP depletion under the conditions of DNA damage also leads to potent PARP trapping. Next, we investigated the effects of these inhibitors on DSB repair. Surprisingly, combination MDV3800/MMS lead to an overall decrease in HR and increase in ALT NHEJ activity. These results were observed in both our BRCA+/- lines. Similar results are observed between the repair activities when using FK866/MMS treatments. Overall, our results suggest that PARP inhibitors with potent PARP trapping activity increases ALT NHEJ, which may potentially compensate for lack of HR. Studies are ongoing to determine whether targeting ALT NHEJ may increase cytotoxicity in these cells. Citation Format: Bryan M. Pelkey, Pratik Nagaria, and Feyruz V. Rassool. PARP trapping by PARP inhibitors have distinct effects on HR and ALT NHEJ DSB repair, potentially impacting its therapeutic efficacy in breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2478. doi:10.1158/1538-7445.AM2017-2478