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Chk1 Requirement for High Global Rates of Replication Fork Progression during Normal Vertebrate S Phase

机译：Chk1对正常脊椎动物S期中复制叉前进的高全局速率的要求

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摘要

Chk1 protein kinase maintains replication fork stability in metazoan cells in response to DNA damage and DNA replication inhibitors. Here, we have employed DNA fiber labeling to quantify, for the first time, the extent to which Chk1 maintains global replication fork rates during normal vertebrate S phase. We report that replication fork rates in Chk1?/? chicken DT40 cells are on average half of those observed with wild-type cells. Similar results were observed if Chk1 was inhibited or depleted in wild-type DT40 cells or HeLa cells by incubation with Chk1 inhibitor or small interfering RNA. In addition, reduced rates of fork extension were observed with permeabilized Chk1?/? cells in vitro. The requirement for Chk1 for high fork rates during normal S phase was not to suppress promiscuous homologous recombination at replication forks, because inhibition of Chk1 similarly slowed fork progression in XRCC3?/? DT40 cells. Rather, we observed an increased number of replication fibers in Chk1?/? cells in which the nascent strand is single-stranded, supporting the idea that slow global fork rates in unperturbed Chk1?/? cells are associated with the accumulation of aberrant replication fork structures.
机译：Chk1蛋白激酶响应DNA损伤和DNA复制抑制剂，在后生细胞中维持复制叉的稳定性。在这里，我们首次使用DNA纤维标记来量化Chk1在正常脊椎动物S期维持整体复制叉率的程度。我们报告说， Chk1 ？/？鸡DT40细胞的复制叉速率平均是野生型细胞观察到的一半。如果通过与Chk1抑制剂或小干扰RNA孵育在野生型DT40细胞或HeLa细胞中抑制或消除了Chk1，则观察到相似的结果。另外，在体外透化的 Chk1 ？/？细胞中观察到了叉子延伸的速率降低。在正常S期中对高分叉率的Chk1的要求并不是要抑制复制分叉的混杂同源重组，因为抑制Chk1同样会减缓 XRCC3 ？/？ DT40细胞。而是，我们观察到新生链为单链的 Chk1 ？/？细胞中复制纤维的数量增加，这支持了慢速全局分叉速率不受干扰的想法 Chk1 ？/？细胞与异常复制叉结构的积累有关。

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