The DNA replication checkpoint response stabilizes stalled replication forks
The DNA replication checkpoint response stabilizes stalled replication forks
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DOI：
10.1038/35087613
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发表时间：
2001-08-02
期刊：
影响因子：
64.8
通讯作者：
Foiani, M
中科院分区：
文献类型：
--
作者：
Lopes, M;Cotta-Ramusino, C;Foiani, M
为了应对DNA损伤和复制阻断，真核生物激活检查点途径，通过协调细胞周期进程与DNA修复来防止基因组不稳定性和癌症（1-5）。在芽殖酵母中，检查点反应需要Mec 1依赖性激活Rad 53蛋白激酶（3，4，6）。当DNA受损时，活性Rad 53会减缓DNA合成（7），并阻止晚期复制起点的启动（8，9）。此外，rad 53突变体不能从复制阻断10恢复。Mec 1和Rad 53还调节不同DNA复制和修复酶的磷酸化状态（6，11 -13）。当DNA被破坏或复制受阻时，检查点通路与复制装置相互作用的机制知之甚少。我们使用二维凝胶技术（14）来检查响应于羟基脲诱导的复制阻断的复制中间体。在这里，我们表明，羟基脲处理的rad 53突变体积累不寻常的DNA结构在复制叉。这些异常分子在从羟基脲阻断恢复期间的持续存在与不能使Rad 53去磷酸化相关。此外，需要Rad 53在块期间正确地维护稳定的复制分叉。我们建议Rad 53在复制暂停时防止分叉崩溃。
In response to DNA damage and blocks to replication, eukaryotes activate the checkpoint pathways that prevent genomic instability and cancer by coordinating cell cycle progression with DNA repair(1-5). In budding yeast, the checkpoint response requires the Mec1-dependent activation of the Rad53 protein kinase(3,4,6). Active Rad53 slows DNA synthesis when DNA is damaged(7) and prevents firing of late origins of replication(8,9). Further, rad53 mutants are unable to recover from a replication block 10. Mec1 and Rad53 also modulate the phosphorylation state of different DNA replication and repair enzymes(6,11-13). Little is known of the mechanisms by which checkpoint pathways interact with the replication apparatus when DNA is damaged or replication blocked. We used the two-dimensional gel technique(14) to examine replication intermediates in response to hydroxyurea-induced replication blocks. Here we show that hydroxyurea-treated rad53 mutants accumulate unusual DNA structures at replication forks. The persistence of these abnormal molecules during recovery from the hydroxyurea block correlates with the inability to dephosphorylate Rad53. Further, Rad53 is required to properly maintain stable replication forks during the block. We propose that Rad53 prevents collapse of the fork when replication pauses.