Extensive RPA2 hyperphosphorylation promotes apoptosis in response to DNA replication stress in CHK1 inhibited cells.
Extensive RPA2 hyperphosphorylation promotes apoptosis in response to DNA replication stress in CHK1 inhibited cells.
复制标题
DOI：
10.1093/nar/gkv835
复制
发表时间：
2015-11-16
影响因子：
14.9
通讯作者：
Meuth M
中科院分区：
文献类型：
--
作者：
Zuazua-Villar P;Ganesh A;Phear G;Gagou ME;Meuth M
在停滞的复制叉处形成的复制蛋白A（RPA）-ssDNA复合物募集关键蛋白以激活ATR-CHK 1信号级联。当CHK 1在DNA复制应激期间被抑制时，RPA 2被广泛过度磷酸化。在这里，我们研究了当CHK 1被抑制时，RPA 2过度磷酸化在细胞命运中的作用。我们发现，通常参与DNA修复（RAD 51）或RPA磷酸化（PP 4蛋白磷酸酶复合物）的控制蛋白质在CHK 1和DNA合成抑制剂处理后不会被招募到基因组中。这不是由于RPA 2过度磷酸化，因为抑制这种反应不会恢复负载，表明募集需要活性CHK 1。为了确定RPA 2过度磷酸化是否保护停滞的叉在复制应激期间免于崩溃或诱导CHK 1抑制的细胞中的凋亡，表征了表达在关键磷酸化位点突变的RPA 2基因的细胞。突变RPA 2拯救细胞免于RPA 2耗尽，并降低CHK 1和复制抑制剂处理诱导的细胞凋亡水平，但双链断裂的发生率不受影响。我们的数据表明，RPA 2过度磷酸化促进细胞死亡，在复制应激时，CHK 1功能受损，但似乎并不是必不可少的复制叉的完整性。
The replication protein A (RPA)–ssDNA complex formed at arrested replication forks recruits key proteins to activate the ATR-CHK1 signalling cascade. When CHK1 is inhibited during DNA replication stress, RPA2 is extensively hyperphosphorylated. Here, we investigated the role of RPA2 hyperphosphorylation in the fate of cells when CHK1 is inhibited. We show that proteins normally involved in DNA repair (RAD51) or control of RPA phosphorylation (the PP4 protein phosphatase complex) are not recruited to the genome after treatment with CHK1 and DNA synthesis inhibitors. This is not due to RPA2 hyperphosphorylation as suppression of this response does not restore loading suggesting that recruitment requires active CHK1. To determine whether RPA2 hyperphosphorylation protects stalled forks from collapse or induction of apoptosis in CHK1 inhibited cells during replication stress, cells expressing RPA2 genes mutated at key phosphorylation sites were characterized. Mutant RPA2 rescued cells from RPA2 depletion and reduced the level of apoptosis induced by treatment with CHK1 and replication inhibitors however the incidence of double strand breaks was not affected. Our data indicate that RPA2 hyperphosphorylation promotes cell death during replication stress when CHK1 function is compromised but does not appear to be essential for replication fork integrity.