DNA Replication Checkpoint Signaling Depends on a Rad53-Dbf4 N-Terminal Interaction in Saccharomyces cerevisiae
DNA Replication Checkpoint Signaling Depends on a Rad53-Dbf4 N-Terminal Interaction in Saccharomyces cerevisiae
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DOI：
10.1534/genetics.113.149740
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发表时间：
2013-06-01
期刊：
影响因子：
3.3
通讯作者：
Weinreich, Michael
中科院分区：
文献类型：
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作者：
Chen, Ying-Chou;Kenworthy, Jessica;Weinreich, Michael
Dbf 4依赖性激酶（DDK）和细胞周期蛋白依赖性激酶（CDK）是启动DNA复制在各个起点所必需的。在复制应激期间，S期检查点抑制晚期复制起点的DDK和CDK依赖性激活。Rad 53激酶是复制检查点的中心效应子，并且结合并磷酸化Dbf 4以防止晚起源放电。Rad 53-Dbf 4物理相互作用的分子基础尚不清楚，但通过Dbf 4 N末端发生。在这里，我们发现Rad 53 FHA 1和FHA 2结构域，特异性识别磷酸苏氨酸（pT），通过N-末端序列和相邻的BRCT结构域与Dbf 4相互作用。纯化的Rad 53 FHA 1结构域（而不是FHA 2）结合到pT Dbf 4肽在体外，这表明可能的磷酸-苏氨酸依赖的FHA 1和Dbf 4之间的相互作用。Dbf 4-Rad 53相互作用受Dbf 4 N末端中Cdc 5-和Msa 1-结合位点可分离的多个接触控制。重要的是，Rad 53-Dbf 4物理相互作用的消除阻断了Dbf 4磷酸化，并允许复制检查点激活期间的晚起源放电。这表明Rad 53必须稳定地结合Dbf 4以调节其活性。
Dbf4-dependent kinase (DDK) and cyclin-dependent kinase (CDK) are essential to initiate DNA replication at individual origins. During replication stress, the S-phase checkpoint inhibits the DDK- and CDK-dependent activation of late replication origins. Rad53 kinase is a central effector of the replication checkpoint and both binds to and phosphorylates Dbf4 to prevent late-origin firing. The molecular basis for the Rad53-Dbf4 physical interaction is not clear but occurs through the Dbf4 N terminus. Here we found that both Rad53 FHA1 and FHA2 domains, which specifically recognize phospho-threonine (pT), interacted with Dbf4 through an N-terminal sequence and an adjacent BRCT domain. Purified Rad53 FHA1 domain (but not FHA2) bound to a pT Dbf4 peptide in vitro, suggesting a possible phospho-threonine-dependent interaction between FHA1 and Dbf4. The Dbf4-Rad53 interaction is governed by multiple contacts that are separable from the Cdc5- and Msa1-binding sites in the Dbf4 N terminus. Importantly, abrogation of the Rad53-Dbf4 physical interaction blocked Dbf4 phosphorylation and allowed late-origin firing during replication checkpoint activation. This indicated that Rad53 must stably bind to Dbf4 to regulate its activity.