Molecular and Computational Biology, University of Southern California, Los Angeles, California, USA.
Mol Cell Biol. 2012 Dec;32(24):4986-97. doi: 10.1128/MCB.01060-12. Epub 2012 Oct 8.
Hydroxyurea (HU) treatment activates the intra-S phase checkpoint proteins Cds1 and Mrc1 to prevent replication fork collapse. We found that prolonged DNA synthesis occurs in cds1Δ and mrc1Δ checkpoint mutants in the presence of HU and continues after release. This is coincident with increased DNA damage measured by phosphorylated histone H2A in whole cells during release. High-resolution live-cell imaging shows that mutants first accumulate extensive replication protein A (RPA) foci, followed by increased Rad52. Both DNA synthesis and RPA accumulation require the MCM helicase. We propose that a replication fork "collapse point" in HU-treated cells describes the point at which accumulated DNA damage and instability at individual forks prevent further replication. After this point, cds1Δ and mrc1Δ forks cannot complete genome replication. These observations establish replication fork collapse as a dynamic process that continues after release from HU block.
羟基脲 (HU) 处理激活细胞内 S 期检查点蛋白 Cds1 和 Mrc1，以防止复制叉崩溃。我们发现，在 HU 存在的情况下，cds1Δ 和 mrc1Δ 检查点突变体中会发生长时间的 DNA 合成，并且在释放后仍会继续。这与释放过程中整个细胞中磷酸化组蛋白 H2A 测量到的增加的 DNA 损伤相一致。高分辨率活细胞成像显示，突变体首先积累大量的复制蛋白 A (RPA) 焦点，随后 Rad52 增加。DNA 合成和 RPA 积累都需要 MCM 解旋酶。我们提出，HU 处理细胞中的复制叉“崩溃点”描述了在单个叉上积累的 DNA 损伤和不稳定性阻止进一步复制的点。在此之后，cds1Δ 和 mrc1Δ 叉无法完成基因组复制。这些观察结果确立了复制叉崩溃作为一个动态过程，在 HU 阻断释放后仍会继续。
