Claspin Promotes Normal Replication Fork Rates in Human Cells

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Abstract
The S phase-specific adaptor protein Claspin mediates the checkpoint response to replication stress by facilitating phosphorylation of Chk1 by ataxia-telangiectasia and Rad3-related (ATR). Evidence suggests that these components of the ATR pathway also play a critical role during physiological S phase. Chk1 is required for high rates of global replication fork progression, and Claspin interacts with the replication machinery and might therefore monitor normal DNA replication. Here, we have used DNA fiber labeling to investigate, for the first time, whether human Claspin is required for high rates of replication fork progression during normal S phase. We report that Claspin-depleted HeLa and HCT116 cells display levels of replication fork slowing similar to those observed in Chk1-depleted cells. This was also true in primary human 1BR3 fibroblasts, albeit to a lesser extent, suggesting that Claspin is a universal requirement for high replication fork rates in human cells. Interestingly, Claspin-depleted cells retained significant levels of Chk1 phosphorylation at both Ser317 and Ser345, raising the possibility that Claspin function during normal fork progression may extend beyond facilitating phosphorylation of either individual residue. Consistent with this possibility, depletion of Chk1 and Claspin together doubled the percentage of very slow forks, compared with depletion of either protein alone.