Cdc45 is limiting for replication initiation in humans

Cdc45 is an essential protein that together with Mcm2-7 and GINS forms the eukaryotic replicative helicase CMG. Cdc45 seems to be rate limiting for the initial unwinding or firing of replication origins. In line with this view, Cdc45-overexpressing cells fired at least twice as many origins as control cells. However, these cells displayed an about 2-fold diminished fork elongation rate, a pronounced asymmetry of replication fork extension, and an early S phase arrest. This was accompanied by H2AX-phosphorylation and subsequent apoptosis. Unexpectedly, we did not observe increased ATR/Chk1 signaling but rather a mild ATM/Chk2 response. In addition, we detected accumulation of long stretches of single-stranded DNA, a hallmark of replication catastrophe. We conclude that increased origin firing by upregulated Cdc45 caused exhaustion of the single-strand binding protein RPA, which in consequence diminished the ATR/Chk1 response; the subsequently occurring fork breaks led to an ATM/Chk2 mediated phosphorylation of H2AX and eventually to apoptosis.

Cdc45（细胞分裂周期蛋白45）是一类必需蛋白，可与Mcm2-7（微型染色体维持蛋白2-7）及GINS复合物共同组成真核生物复制性解旋酶CMG。Cdc45似乎是复制原点初始解旋与激活的限速因子。与此观点一致，过表达Cdc45的细胞中被激活的复制原点数量至少为对照细胞的两倍。然而，此类细胞的复制叉延伸速率降低约一倍，复制叉延伸呈现显著不对称性，并出现早期S期阻滞。上述现象伴随H2AX磷酸化及后续细胞凋亡的发生。出乎意料的是，本研究未检测到ATR/Chk1信号通路的激活增强，反而仅出现轻度的ATM/Chk2应答反应。此外，研究还检测到长片段单链DNA的积累——这是复制灾难的标志性特征。综上，Cdc45表达上调引发复制原点激活增多，进而导致单链结合蛋白RPA耗竭，最终削弱了ATR/Chk1信号通路的应答；随后发生的复制叉断裂通过ATM/Chk2通路介导H2AX磷酸化，最终引发细胞凋亡。