Sánchez Arancha, Roguev Assen, Krogan Nevan J, Russell Paul
Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California 92037.
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94158.
G3 (Bethesda). 2015 Mar 19;5(5):953-62. doi: 10.1534/g3.115.017251.
Brc1, which was first identified as a high-copy, allele-specific suppressor of a mutation impairing the Smc5-Smc6 holocomplex in Schizosaccharomyces pombe, protects genome integrity during normal DNA replication and when cells are exposed to toxic compounds that stall or collapse replication forks. The C-terminal tandem BRCT (BRCA1 C-terminus) domain of fission yeast Brc1 docks with phosphorylated histone H2A (γH2A)-marked chromatin formed by ATR/Rad3 checkpoint kinase at arrested and damaged replication forks; however, how Brc1 functions in relation to other genome protection modules remains unclear. Here, an epistatic mini-array profile reveals critical requirements for Brc1 in mutants that are defective in multiple DNA damage response pathways, including checkpoint signaling by Rad3-Rad26/ATR-ATRIP kinase, DNA repair by Smc5-Smc6 holocomplex, replication fork stabilization by Mrc1/claspin and Swi1-Swi3/Timeless-Tipin, and control of ubiquitin-regulated proteolysis by the COP9 signalosome (CSN). Exogenous genotoxins enhance these negative genetic interactions. Rad52 and RPA foci are increased in CSN-defective cells, and loss of γH2A increases genotoxin sensitivity, indicating a critical role for the γH2A-Brc1 module in stabilizing replication forks in CSN-defective cells. A negative genetic interaction with the Nse6 subunit of Smc5-Smc6 holocomplex indicates that the DNA repair functions of Brc1 and Smc5-Smc6 holocomplex are at least partially independent. Rtt107, the Brc1 homolog in Saccharomyces cerevisiae, has a very different pattern of genetic interactions, indicating evolutionary divergence of functions and DNA damage responses.
Brc1最初被鉴定为粟酒裂殖酵母中一个高拷贝、等位基因特异性的抑制子，可抑制损害Smc5 - Smc6全复合体的突变，在正常DNA复制过程中以及细胞暴露于使复制叉停滞或崩溃的有毒化合物时，保护基因组完整性。裂殖酵母Brc1的C端串联BRCT（BRCA1 C端）结构域与由ATR/Rad3检查点激酶在停滞和受损复制叉处形成的磷酸化组蛋白H2A（γH2A）标记的染色质对接；然而，Brc1与其他基因组保护模块相关的功能仍不清楚。在这里，上位性微阵列分析揭示了在多个DNA损伤反应途径存在缺陷的突变体中对Brc1的关键需求，这些途径包括Rad3 - Rad26/ATR - ATRIP激酶的检查点信号传导、Smc5 - Smc6全复合体的DNA修复、Mrc1/claspin和Swi1 - Swi3/Timeless - Tipin对复制叉的稳定作用以及COP9信号体（CSN）对泛素调节的蛋白水解的控制。外源性基因毒素会增强这些负向遗传相互作用。在CSN缺陷细胞中Rad52和RPA病灶增加，γH2A的缺失会增加基因毒素敏感性，表明γH2A - Brc1模块在稳定CSN缺陷细胞中的复制叉方面起关键作用。与Smc5 - Smc6全复合体的Nse6亚基的负向遗传相互作用表明，Brc1和Smc5 - Smc6全复合体的DNA修复功能至少部分独立。酿酒酵母中Brc1的同源物Rtt107具有非常不同的遗传相互作用模式，表明功能和DNA损伤反应存在进化差异。
