Kimble Michael T, Sane Aakanksha, Reid Robert J D, Johnson Matthew J, Rothstein Rodney, Symington Lorraine S
Program in Biological Sciences, Columbia University, New York, NY 10027, USA; Department of Microbiology & Immunology, Columbia University Irving Medical Center, New York, NY 10032, USA.
Department of Microbiology & Immunology, Columbia University Irving Medical Center, New York, NY 10032, USA.
Mol Cell. 2025 Jan 2;85(1):61-77.e6. doi: 10.1016/j.molcel.2024.10.032. Epub 2024 Dec 3.
Single-strand breaks (SSBs) are one of the most commonly occurring endogenous lesions with the potential to give rise to cytotoxic double-strand breaks (DSBs) during DNA replication. To investigate how replication-dependent DSBs are repaired, we employed Cas9 nickase (nCas9) to generate site- and strand-specific nicks in the budding yeast genome. We found that nCas9-induced nicks are converted to mostly double-ended DSBs during S phase. Repair of replication-associated DSBs requires homologous recombination (HR) and is independent of classical non-homologous end joining. Consistent with a strong bias to repair these lesions using a sister-chromatid template, we observed minimal induction of inter-chromosomal HR by nCas9. In a genome-wide screen to identify factors necessary for the repair of replication-dependent DSBs, we recovered components of the replication-coupled nucleosome assembly (RCNA) pathway. Our findings suggest that the RCNA pathway is especially important to repair DSBs arising from nicks in the leading-strand template through acetylation of histone H3K56.
单链断裂（SSB）是最常见的内源性损伤之一，在DNA复制过程中有可能引发细胞毒性双链断裂（DSB）。为了研究复制依赖性DSB是如何修复的，我们利用Cas9切口酶（nCas9）在芽殖酵母基因组中产生位点和链特异性切口。我们发现，nCas9诱导的切口在S期大多会转化为双端DSB。复制相关DSB的修复需要同源重组（HR），且独立于经典的非同源末端连接。与使用姐妹染色单体模板修复这些损伤的强烈偏好一致，我们观察到nCas9对染色体间HR的诱导作用极小。在全基因组筛选以确定复制依赖性DSB修复所需的因子时，我们筛选到了复制偶联核小体组装（RCNA）途径的成分。我们的研究结果表明，RCNA途径对于通过组蛋白H3K56乙酰化修复前导链模板切口中产生的DSB尤为重要。
