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Recruitment of DNA repair proteins to DNA damage sites is a critical step for DNA repair. Post-translational modifications of proteins at DNA damage sites serve as DNA damage codes to recruit specific DNA repair factors. Here, we show that mRNA is locally modified by m 5 C at sites of DNA damage. The RNA methyltransferase TRDMT1 is recruited to DNA damage sites to promote m 5 C induction. Loss of TRDMT1 compromises homologous recombination (HR) and increases cellular sensitivity to DNA double-strand breaks (DSBs). In the absence of TRDMT1, RAD51 and RAD52 fail to localize to sites of reactive oxygen species (ROS)-induced DNA damage. In vitro, RAD52 displays an increased affinity for DNA:RNA hybrids containing m 5 C-modified RNA. Loss of TRDMT1 in cancer cells confers sensitivity to PARP inhibitors in vitro and in vivo. These results reveal an unexpected TRDMT1-m 5 C axis that promotes HR, suggesting that post-transcriptional modifications of RNA can also serve as DNA damage codes to regulate DNA repair. Post-translational modifications of proteins at DNA damage sites can facilitate the recruitment of DNA repair factors. Here, the authors show that mRNA is locally modified with m 5 C at sites of DNA damage by the RNA methyltransferase TRDMT1 to promote homologous recombination repair.

Damage repair mechanisms at transcriptionally active sites during the G0/G1 phase are largely unknown. To elucidate these mechanisms, we introduced genome site-specific oxidative DNA damage and determined the role of transcription in repair factor assembly. We find that KU and NBS1 are recruited to damage sites independent of transcription. However, assembly of RPA1, RAD51C, RAD51, and RAD52 at such sites is strictly governed by active transcription and requires both wild-type Cockayne syndrome protein B (CSB) function and the presence of RNA in the G0/G1 phase. We show that the ATPase activity of CSB is indispensable for loading and binding of the recombination factors. CSB counters radiation-induced DNA damage in both cells and zebrafish models. Taken together, our results have uncovered a novel, RNA-based recombination mechanism by which CSB protects genome stability from strand breaks at transcriptionally active sites and may provide insight into the clinical manifestations of Cockayne syndrome.

Non-homologous end joining (NHEJ) is a major pathway to repair DNA double-strand breaks (DSBs), which can display different types of broken ends. However, it is unclear how NHEJ factors organize to repair diverse types of DNA breaks. Here, through systematic analysis of the human NHEJ factor interactome, we identify PAXX as a direct interactor of Ku. The crystal structure of PAXX is similar to those of XRCC4 and XLF. Importantly, PAXX-deficient cells are sensitive to DSB-causing agents. Moreover, epistasis analysis demonstrates that PAXX functions together with XLF in response to ionizing radiation-induced complex DSBs, whereas they function redundantly in response to Topo2 inhibitor-induced simple DSBs. Consistently, PAXX and XLF coordinately promote the ligation of complex but not simple DNA ends in vitro . Altogether, our data identify PAXX as a new NHEJ factor and provide insight regarding the organization of NHEJ factors responding to diverse types of DSB ends. DNA double-strand breaks (DSBs), a highly deleterious form of DNA damage, are associated with multiple types of broken ends. Here, the authors identify a XRCC4-like factor that functions in the non-homologous end-joining DNA repair pathway to repair DSBs with complex broken ends.

During the DNA damage response (DDR), ubiquitination plays an important role in the recruitment and regulation of repair proteins. However, little is known about elimination of the ubiquitination signal after repair is completed. Here we show that the ubiquitin-specific protease 5 (USP5), a deubiquitinating enzyme, is involved in the elimination of the ubiquitin signal from damaged sites and is required for efficient DNA double-strand break (DSB) repair. Depletion of USP5 sensitizes cells to DNA damaging agents, produces DSBs, causes delayed disappearance of γH2AX foci after Bleocin treatment, and influences DSB repair efficiency in the homologous recombination pathway but not in the non-homologous end joining pathway. USP5 co-localizes to DSBs induced by laser micro-irradiation in a RAD18-dependent manner. Importantly, polyubiquitin chains at sites of DNA damage remained for longer periods in USP5-depleted cells. Our results show that disassembly of polyubiquitin chains by USP5 at sites of damage is important for efficient DSB repair.

BRCA1 is an important gene involved in susceptibility to breast and ovarian cancer and its product regulates the cellular response to DNA double‐strand breaks. Here, we present evidence that BRCA1 also contributes to the transcription‐coupled repair (TCR) of ultraviolet (UV) light‐induced DNA damage. BRCA1 immediately accumulates at the sites of UV irradiation‐mediated damage in cell nuclei in a manner that is fully dependent on both Cockayne syndrome B (CSB) protein and active transcription. Suppression of BRCA1 expression inhibits the TCR of UV lesions and increases the UV sensitivity of cells proficient in TCR. BRCA1 physically interacts with CSB protein. BRCA1 polyubiquitinates CSB and this polyubiquitination and subsequent degradation of CSB occur following UV irradiation, even in the absence of Cockayne syndrome A (CSA) protein. The depletion of BRCA1 expression increases the UV sensitivity of CSA‐deficient cells. These results indicate that BRCA1 is involved in TCR and that a BRCA1‐dependent polyubiquitination pathway for CSB exists alongside the CSA‐dependent pathway to yield more efficient excision repair of lesions on the transcribed DNA strand. (Cancer Sci 2011; 102: 1840–1847)

Ataxia telangiectasia mutated （ATM） mediates DNA damage response by controling irradiation-induced foci formation, cell cycle checkpoint, and apoptosis. However, how upstream signaling regulates ATM is not completely understood. Here, we show that upon irradiation stimulation, ATM associates with and is phosphorylated by epidermal growth factor receptor （EGFR） at Tyr370 （Y370） at the site of DNA double-strand breaks. Depletion of endogenous EGFR impairs ATM-mediated foei formation, homologous recombination, and DNA repair. Moreover, pretreatment with an EGFR kinase inhibitor, gefitinib, blocks EGFR and ATM association, hinders CHK2 activation and subsequent foci formation, and increases radiosensitivity. Thus, we reveal a critical mechanism by which EGFR directly regulates ATM activation in DNA damage response, and our results suggest that the status of ATM Y370 phosphorylation has the potential to serve as a biomarker to stratify patients for either radiotherapy alone or in combination with EGFR inhibition.

Cellular DNA repair processes are crucial to maintain genome stability and integrity. In DNA base excision repair, a tight heterodimer complex formed by DNA polymerase β (Polβ) and XRCC1 is thought to facilitate repair by recruiting Polβ to DNA damage sites. Here we show that disruption of the complex does not impact DNA damage response or DNA repair. Instead, the heterodimer formation is required to prevent ubiquitylation and degradation of Polβ. In contrast, the stability of the XRCC1 monomer is protected from CHIP-mediated ubiquitylation by interaction with the binding partner HSP90. In response to cellular proliferation and DNA damage, proteasome and HSP90-mediated regulation of Polβ and XRCC1 alters the DNA repair complex architecture. We propose that protein stability, mediated by DNA repair protein complex formation, functions as a regulatory mechanism for DNA repair pathway choice in the context of cell cycle progression and genome surveillance. DNA repair is crucial to maintain cellular genome stability and integrity. Here, the authors show that XRCC1 and DNA polymerase β, two essential proteins in DNA repair, heterodimerize to protect the latter from proteasome degradation.

Recruitment of DNA repair proteins to DNA damage sites is a critical step for DNA repair. Post-translational modifications of proteins at DNA damage sites serve as DNA damage codes to recruit specific DNA repair factors. Here, we show that mRNA is locally modified by m C at sites of DNA damage. The RNA methyltransferase TRDMT1 is recruited to DNA damage sites to promote m C induction. Loss of TRDMT1 compromises homologous recombination (HR) and increases cellular sensitivity to DNA double-strand breaks (DSBs). In the absence of TRDMT1, RAD51 and RAD52 fail to localize to sites of reactive oxygen species (ROS)-induced DNA damage. In vitro, RAD52 displays an increased affinity for DNA:RNA hybrids containing m C-modified RNA. Loss of TRDMT1 in cancer cells confers sensitivity to PARP inhibitors in vitro and in vivo. These results reveal an unexpected TRDMT1-m C axis that promotes HR, suggesting that post-transcriptional modifications of RNA can also serve as DNA damage codes to regulate DNA repair.