Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, United Kingdom.
Chemistry Department, School of Life Sciences, University of Sussex, Falmer, United Kingdom.
Elife. 2021 Apr 16;10:e68579. doi: 10.7554/eLife.68579.
The essential Smc5/6 complex is required in response to replication stress and is best known for ensuring the fidelity of homologous recombination. Using single-molecule tracking in live fission yeast to investigate Smc5/6 chromatin association, we show that Smc5/6 is chromatin associated in unchallenged cells and this depends on the non-SMC protein Nse6. We define a minimum of two Nse6-dependent sub-pathways, one of which requires the BRCT-domain protein Brc1. Using defined mutants in genes encoding the core Smc5/6 complex subunits, we show that the Nse3 double-stranded DNA binding activity and the arginine fingers of the two Smc5/6 ATPase binding sites are critical for chromatin association. Interestingly, disrupting the single-stranded DNA (ssDNA) binding activity at the hinge region does not prevent chromatin association but leads to elevated levels of gross chromosomal rearrangements during replication restart. This is consistent with a downstream function for ssDNA binding in regulating homologous recombination.
必需的 Smc5/6 复合物是响应复制应激所必需的，其最主要的功能是确保同源重组的保真度。我们使用活裂殖酵母中的单分子追踪来研究 Smc5/6 与染色质的关联，结果表明 Smc5/6 在未受挑战的细胞中与染色质相关联，这依赖于非 SMC 蛋白 Nse6。我们定义了至少两种依赖于 Nse6 的亚途径，其中一种途径需要 BRCT 结构域蛋白 Brc1。我们使用编码核心 Smc5/6 复合物亚基的基因突变体进行研究，结果表明 Nse3 的双链 DNA 结合活性和两个 Smc5/6 ATP 酶结合位点的精氨酸指对于染色质关联至关重要。有趣的是，破坏铰链区域的单链 DNA（ssDNA）结合活性不会阻止染色质关联，但会导致在复制重新启动期间高水平的染色体结构重排。这与 ssDNA 结合在调节同源重组中的下游功能一致。
