Role of Elg1 protein in double strand break repair

The inaccurate repair of DNA double-strand breaks (DSBs) can result in genomic instability, and additionally cell death or the development of cancer. Elg1, which forms an alternative RFC-like complex with RFC2-5, is required for the maintenance of genome stability in Saccharomyces cerevisiae, and its function has been linked to DNA replication or damage checkpoint response. Here, we show that Elg1 is involved in homologous recombination (HR)-mediated DSB repair. Mutants of elg1 were partially defective in HR induced by methylmethanesufonate (MMS) and phleomycin. Deletion of ELG1 resulted in less efficient repair of phleomycin-induced DSBs in G(2)/M phase-arrested cells. During HR between MAT and HML loci, Elg1 associated with both the MAT locus near the HO endonuclease-induced DSB site, and the HML homologous donor locus. The association of Elg1 with the MAT locus was not dependent on Rad52. However, Elg1 association with the HML locus depended on Rad52. Importantly, we found that two of the later steps in HR-mediated repair of an HO endonuclease-induced DSB, primer extension after strand invasion and ligation, were less efficient in elg1 mutants. Our results suggest that Elg1 is involved in DSB repair by HR

Dpb11, the budding yeast homolog of TopBP1, functions with the checkpoint clamp in recombination repair

Dpb11 is required for the loading of DNA polymerases α and ɛ on to DNA in chromosomal DNA replication and interacts with the DNA damage checkpoint protein Ddc1 in Saccharomyces cerevisiae. The interaction between the homologs of Dpb11 and Ddc1 in human cells and fission yeast is thought to reflect their involvement in the checkpoint response. Here we show that dpb11-1 cells, carrying a mutated Dpb11 that cannot interact with Ddc1, are defective in the repair of methyl methanesulfonate (MMS)-induced DNA damage but not in the DNA damage checkpoint at the permissive temperature. Epistatic analyses suggested that Dpb11 is involved in the Rad51/Rad52-dependent recombination pathway. Ddc1 as well as Dpb11 were required for homologous recombination induced by MMS. Moreover, we found the in vivo association of Dpb11 and Ddc1 with not only the HO-induced double-strand break (DSB) site at MAT locus but also the donor sequence HML during homologous recombination between MAT and HML. Rad51 was required for their association with the HML donor locus, but not with DSB site at the MAT locus. In addition, the association of Dpb11 with the MAT and HML locus after induction of HO-induced DSB was dependent on Ddc1. These results indicate that, besides the involvement in the replication and checkpoint, Dpb11 functions with Ddc1 in the recombination repair process itself

Novel Calcium-Binding Ablating Mutations Induce Constitutive RET Activity and Drive Tumorigenesis

がんゲノム医療のさらなる拡大へ向けた一歩 --コンピュータ解析で意義不明変異のなかに治療標的となる新たな遺伝子変異を発見--. 京都大学プレスリリース. 2022-09-29.Distinguishing oncogenic mutations from variants of unknown significance (VUS) is critical for precision cancer medicine. Here, computational modeling of 71, 756 RET variants for positive selection together with functional assays of 110 representative variants identified a three-dimensional cluster of VUSs carried by multiple human cancers that cause amino acid substitutions in the calmodulin-like motif (CaLM) of RET. Molecular dynamics simulations indicated that CaLM mutations decrease interactions between Ca²⁺ and its surrounding residues and induce conformational distortion of the RET cysteine-rich domain containing the CaLM. RET-CaLM mutations caused ligand-independent constitutive activation of RET kinase by homodimerization mediated by illegitimate disulfide bond formation. RET-CaLM mutants possessed oncogenic and tumorigenic activities that could be suppressed by tyrosine kinase inhibitors targeting RET. This study identifies calcium-binding ablating mutations as a novel type of oncogenic mutation of RET and indicates that in silico–driven annotation of VUSs of druggable oncogenes is a promising strategy to identify targetable driver mutations

Ctf18 is required for homologous recombination-mediated double-strand break repair

The efficient repair of double-strand breaks (DSBs) is crucial in maintaining genomic integrity. Sister chromatid cohesion is important for not only faithful chromosome segregation but also for proper DSB repair. During DSB repair, the Smc1–Smc3 cohesin complex is loaded onto chromatin around the DSB to support recombination-mediated DSB repair. In this study, we investigated whether Ctf18, a factor implicated in the establishment of sister chromatid cohesion, is involved in DSB repair in budding yeast. Ctf18 was recruited to HO-endonuclease induced DSB sites in an Mre11-dependent manner and to damaged chromatin in G2/M phase-arrested cells. The ctf18 mutant cells showed high sensitivity to DSB-inducible genotoxic agents and defects in DSB repair, as well as defects in damage-induced recombination between sister chromatids and between homologous chromosomes. These results suggest that Ctf18 is involved in damage-induced homologous recombination

Novel function of HATs and HDACs in homologous recombination through acetylation of human RAD52 at double-strand break sites

The p300 and CBP histone acetyltransferases are recruited to DNA double-strand break (DSB) sites where they induce histone acetylation, thereby influencing the chromatin structure and DNA repair process. Whether p300/CBP at DSB sites also acetylate non-histone proteins, and how their acetylation affects DSB repair, remain unknown. Here we show that p300/CBP acetylate RAD52, a human homologous recombination (HR) DNA repair protein, at DSB sites. Using in vitro acetylated RAD52, we identified 13 potential acetylation sites in RAD52 by a mass spectrometry analysis. An immunofluorescence microscopy analysis revealed that RAD52 acetylation at DSBs sites is counteracted by SIRT2- and SIRT3-mediated deacetylation, and that non-acetylated RAD52 initially accumulates at DSB sites, but dissociates prematurely from them. In the absence of RAD52 acetylation, RAD51, which plays a central role in HR, also dissociates prematurely from DSB sites, and hence HR is impaired. Furthermore, inhibition of ataxia telangiectasia mutated (ATM) protein by siRNA or inhibitor treatment demonstrated that the acetylation of RAD52 at DSB sites is dependent on the ATM protein kinase activity, through the formation of RAD52, p300/CBP, SIRT2, and SIRT3 foci at DSB sites. Our findings clarify the importance of RAD52 acetylation in HR and its underlying mechanism

Ctf18 is required for homologous recombination-mediated double-strand

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Human SIRT2 and SIRT3 deacetylases function in DNA homologous recombinational repair

SIRT2 and SIRT3 protein deacetylases maintain genome integrity and stability. However, their mechanisms for maintaining the genome remain unclear. To examine the roles of SIRT2 and SIRT3 in DSB repair, I‐SceI‐based GFP reporter assays for HR, single‐strand annealing (SSA), and non‐homologous end joining (NHEJ) repair were performed under SIRT2‐ or SIRT3‐depleted conditions. SIRT2 or SIRT3 depletion inhibited HR repair equally to RAD52 depletion, but did not affect SSA and NHEJ repairs. SIRT2 or SIRT3 depletion disturbed the recruitment of RAD51 to DSB sites, an essential step for RAD51‐dependent HR repair, but not directly through RAD52 deacetylation. SIRT2 or SIRT3 depletion decreased the colocalization of γH2AX foci with RPA1, and thus they might be involved in initiating DSB end resection for the recruitment of RAD51 to DSB sites at an early step in HR repair. These results reveal the novel underlying mechanism of the SIRT2 and SIRT3 functions in HR for genome stability

Rmi1, a member of the Sgs1–Top3 complex in budding yeast, contributes to sister chromatid cohesion

The Saccharomyces cerevisiae RecQ-mediated genome instability (Rmi1) protein was recently identified as the third member of the slow growth suppressor 1–DNA topoisomerase III (Sgs1–Top3) complex, which is required for maintaining genomic stability. Here, we show that cells lacking RMI1 have a mitotic delay, which is partly dependent on the spindle checkpoint, and are sensitive to the microtubule depolymerizing agent benomyl. We show that rmi1 and top3 single mutants are defective in sister chromatid cohesion, and that deletion of SGS1 suppresses benomyl sensitivity and the cohesion defect in these mutant cells. Loss of RAD51 also suppresses the cohesion defect of rmi1 mutant cells. These results indicate the existence of a new pathway involving Rad51 and Sgs1-Top3-Rmi1, which leads to the establishment of sister chromatid cohesion