Kumar Sanjay, Huberman Joel A
Department of Cancer Biology, Roswell Park Cancer Institute, Elm & Carlton Streets, Buffalo, New York 14263-0001, USA.
Mol Cell Biol. 2009 Jan;29(2):602-11. doi: 10.1128/MCB.01319-08. Epub 2008 Nov 10.
To elucidate the checkpoint mechanism responsible for slowing passage through S phase when fission yeast cells are treated with the DNA-damaging agent methyl methanesulfonate (MMS), we carried out two-dimensional gel analyses of replication intermediates in cells synchronized by cdc10 block (in G(1)) followed by release into synchronous S phase. The results indicated that under these conditions early-firing centromeric origins were partially delayed but late-firing telomeric origins were not delayed. Replication intermediates persisted in MMS-treated cells, suggesting that replication fork movement was inhibited. These effects were dependent on the Cds1 checkpoint kinase and were abolished in cells overexpressing the Cdc25 phosphatase, suggesting a role for the Cdc2 cyclin-dependent kinase. We conclude that both partial inhibition of the firing of a subset of origins and inhibition of replication fork movement contribute to the slowing of S phase in MMS-treated fission yeast cells.
为了阐明裂殖酵母细胞用DNA损伤剂甲磺酸甲酯（MMS）处理时，导致S期进程减缓的检查点机制，我们对通过cdc10阻断（在G1期）同步化，然后释放到同步S期的细胞中的复制中间体进行了二维凝胶分析。结果表明，在这些条件下，早期激活的着丝粒起始点部分延迟，但晚期激活的端粒起始点没有延迟。复制中间体在MMS处理的细胞中持续存在，表明复制叉移动受到抑制。这些效应依赖于Cds1检查点激酶，并且在过表达Cdc25磷酸酶的细胞中被消除，提示细胞周期蛋白依赖性激酶Cdc2发挥了作用。我们得出结论，起始点子集激活的部分抑制和复制叉移动的抑制都导致了MMS处理的裂殖酵母细胞中S期的减缓。
