Stabilization of stalled replication forks by fork-binding proteins

我们之前已经证明，大肠杆菌的PriA蛋白与RecG合作，可以特异性地结合并稳定受阻的复制叉子。在这项提议中，我们试图通过利用这两种蛋白质来稳定真核细胞中停滞的复制叉子。我们首先开发了一种针对PriA的抗体，并证明了通过芯片分析可以检测到停滞的复制叉。作为对分叉阻断处理的反应，在野生型细胞中，PriA与ORIC结合，而在ORIC不起作用的情况下，它与TERC区结合。然后，我们在分裂酵母细胞中表达了PriA，并检测了它与染色质的结合。PriA在复制起始点附近富含。我们现在正在各种突变细胞中表达PriA，并将通过测量SSB包被的单链的长度来检验PriA是否能够稳定分叉。

We previously showed that E. coli PriA protein, in collaboration with RecG, specifically binds and can stabilize an arrested replication fork. In this proposal we attempted to stabilize stalled replication forks in eukaryotic cells by utilizing these two proteins. We first developed an antibody against PriA and showed that stalled replication forks can be detected by ChIP-chip analyses. In response to fork arrest treatment, PriA bound to oriC in the wild-type cells, whereas it bound to the terC region under the condition where oriC is not functional. We then expressed PriA in fission yeast cells, examined its binding to chromatin. PriA was enriched near the replication origins. We are now expressing PriA in various mutant cells, and will examine if PriA can stabilize the fork by measuring the length of SSB coated single-strand.