Lucca Chiara, Vanoli Fabio, Cotta-Ramusino Cecilia, Pellicioli Achille, Liberi Giordano, Haber James, Foiani Marco
Istituto FIRC di Oncologia Molecolare, Via Adamello 16, Milano 20141, Italy.
Oncogene. 2004 Feb 12;23(6):1206-13. doi: 10.1038/sj.onc.1207199.
The replication checkpoint controls the integrity of replicating chromosomes by stabilizing stalled forks, thus preventing the accumulation of abnormal replication and recombination intermediates that contribute to genome instability. Checkpoint-defective cells are susceptible to rearrangements at chromosome fragile sites when replication pauses, and certain human cancer prone diseases suffer checkpoint abnormalities. It is unclear as to how the checkpoint stabilizes stalled forks and how cells sense replication blocks. We have analysed the checkpoint contribution in controlling replisome-fork association when replication pauses. We show that in yeast wild-type cells, stalled forks exhibit stable replisome complexes and the checkpoint sensors Ddc1 and Ddc2, thus activating Rad53 checkpoint kinase. Ddc1/Ddc2 recruitment on stalled forks and Rad53 activation are influenced by the single-strand-binding protein replication factor A (RFA). rad53 forks exhibit a defective association with DNA polymerases alpha, epsilon and delta. Further, in rad53 mutants, stalled forks progressively generate abnormal structures that turn into checkpoint signals by accumulating RFA, Ddc1 and Ddc2. We suggest that, following replication blocks, checkpoint activation mediated by RFA-ssDNA filaments stabilizes stalled forks by controlling replisome-fork association, thus preventing unscheduled recruitment of recombination enzymes that could otherwise cause the pathological processing of the forks.
复制检查点通过稳定停滞的复制叉来控制复制染色体的完整性，从而防止导致基因组不稳定的异常复制和重组中间体的积累。当复制暂停时，检查点缺陷型细胞在染色体脆弱位点易发生重排，某些人类易患癌症的疾病存在检查点异常。目前尚不清楚检查点如何稳定停滞的复制叉以及细胞如何感知复制阻滞。我们分析了复制暂停时检查点在控制复制体与复制叉关联方面的作用。我们发现，在酵母野生型细胞中，停滞的复制叉呈现出稳定的复制体复合物以及检查点传感器Ddc1和Ddc2，从而激活Rad53检查点激酶。停滞复制叉上Ddc1/Ddc2的募集以及Rad53的激活受单链结合蛋白复制因子A（RFA）的影响。rad53复制叉与DNA聚合酶α、ε和δ的关联存在缺陷。此外，在rad53突变体中，停滞的复制叉逐渐产生异常结构，这些结构通过积累RFA、Ddc1和Ddc2而转化为检查点信号。我们认为，在复制阻滞之后，由RFA-ssDNA细丝介导的检查点激活通过控制复制体与复制叉的关联来稳定停滞的复制叉，从而防止重组酶的意外募集，否则这些重组酶可能导致复制叉的病理性加工。
