Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, UK.
Nature. 2010 Sep 23;467(7314):474-8. doi: 10.1038/nature09373. Epub 2010 Sep 12.
The initiation of eukaryotic DNA replication is regulated by three protein kinase classes: cyclin-dependent kinases (CDK), Dbf4-dependent kinase (DDK) and the DNA damage checkpoint kinases. CDK phosphorylation of two key initiation factors, Sld2 and Sld3, promotes essential interactions with Dpb11 (refs 2-4), whereas DDK acts by phosphorylating subunits of the Mcm2-7 helicase. CDK has an additional role in replication by preventing the re-loading of Mcm2-7 during the S, G2 and M phases, thus preventing origin re-firing and re-replication. During the G1 phase, both CDK and DDK are downregulated, which allows origin licensing and prevents premature replication initiation. Origin firing is also inhibited during the S phase when DNA damage or replication fork stalling activates the checkpoint kinases. Here we show that, analogous to the situation in the G1 phase, the Saccharomyces cerevisiae checkpoint kinase Rad53 inhibits both CDK- and DDK-dependent pathways, which acts redundantly to block further origin firing. Rad53 acts on DDK directly by phosphorylating Dbf4, whereas the CDK pathway is blocked by Rad53-mediated phosphorylation of the downstream CDK substrate, Sld3. This allows CDK to remain active during the S phase in the presence of DNA damage, which is crucial to prevent re-loading of Mcm2-7 onto origins that have already fired. Our results explain how checkpoints regulate origin firing and demonstrate that the slowing of S phase by the 'intra-S checkpoint' is primarily due to the inhibition of origin firing.
真核生物 DNA 复制的起始受三类蛋白激酶调节:细胞周期蛋白依赖性激酶(CDK)、Dbf4 依赖性激酶(DDK)和 DNA 损伤检查点激酶。CDK 对两个关键起始因子 Sld2 和 Sld3 的磷酸化作用促进了与 Dpb11 的必需相互作用(参考文献 2-4),而 DDK 通过磷酸化 Mcm2-7 解旋酶的亚基起作用。CDK 通过在 S、G2 和 M 期防止 Mcm2-7 的再加载来发挥其在复制中的额外作用,从而防止起始原点重新激活和再次复制。在 G1 期,CDK 和 DDK 均下调,从而允许起始原点许可并防止过早的复制起始。当 DNA 损伤或复制叉停滞激活检查点激酶时,起始原点的激活也会受到抑制。在这里,我们表明,类似于 G1 期的情况,酿酒酵母检查点激酶 Rad53 抑制 CDK 和 DDK 依赖性途径,这两种途径协同作用以阻止进一步的起始原点激活。Rad53 通过磷酸化 Dbf4 直接作用于 DDK,而 CDK 途径被 Rad53 介导的下游 CDK 底物 Sld3 的磷酸化所阻断。这使得 CDK 在存在 DNA 损伤的情况下在 S 期保持活性,这对于防止已激活的起始原点重新加载 Mcm2-7 至关重要。我们的结果解释了检查点如何调节起始原点的激活,并表明 S 期的减缓主要是由于起始原点的激活受到抑制。