The Dbf4 motif C zinc finger promotes DNA replication and mediates resistance to genotoxic stress
The Dbf4 motif C zinc finger promotes DNA replication and mediates resistance to genotoxic stress
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DOI：
10.4161/cc.9.10.11752
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发表时间：
2010-05-15
期刊：
影响因子：
4.3
通讯作者：
Duncker, Bernard P.
中科院分区：
文献类型：
--
作者：
Jones, Darryl R.;Prasad, Ajai A.;Duncker, Bernard P.
Dbf4/CDC7激酶(DDK)通过磷酸化McM2-7解旋酶复合体的亚基，在刺激DNA复制中起着至关重要的作用。当S阶段检查点被触发时，这种激酶复合体本身被磷酸化，并以依赖于RAD53的方式从染色质中移除。对各种真核生物的Dbf4序列进行比较，发现了三个保守的区域，分别称为基序N、M和C。其中最保守的基序C编码一个锌指，已知参与蛋白质-蛋白质和蛋白质-DNA的相互作用。保守基序C半胱氨酸和组氨酸的突变破坏了Dbf4与ARS1起源DNA和McM2的结合，但不破坏其他已知的配体，包括CDC7、Rad53或起源识别复合体Orc2。此外，这些突变还削弱了Dbf4磷酸化Mcm2的能力。用这些基序C突变等位基因替换单个基因组DBF4拷贝的萌芽酵母菌株进入并进入S期，表明观察到的McM2相互作用的减弱阻止了DDK有效地刺激DNA复制的启动。在启动后，McM2-7随复制叉子迁移。有趣的是，基序C突变体对长期暴露于基因毒剂羟基脲和甲磺酸甲酯敏感，而不是短期暴露。这些结果支持这样一个模型，即在触发延长的S阶段检查点的条件下，DDK与McM2的相互作用对于稳定和/或重新启动复制分叉是重要的。
The Dbf4/Cdc7 kinase (DDK) plays an essential role in stimulating DNA replication by phosphorylating subunits of the Mcm2-7 helicase complex at origins. This kinase complex is itself phosphorylated and removed from chromatin in a Rad53-dependent manner when an S phase checkpoint is triggered. Comparison of Dbf4 sequence across a variety of eukaryotic species has revealed three conserved regions that have been termed motifs N, M and C. The most highly conserved of the three, motif C, encodes a zinc finger, which are known to mediate protein-protein and protein-DNA interactions. Mutation of conserved motif C cysteines and histidines disrupted the association of Dbf4 with ARS1 origin DNA and Mcm2, but not other known ligands including Cdc7, Rad53 or the origin recognition complex subunit Orc2. Furthermore, these mutations impaired the ability of Dbf4 to phosphorylate Mcm2. Budding yeast strains for which the single genomic DBF4 copy was replaced with these motif C mutant alleles were compromised for entry into and progression through S phase, indicating that the observed weakening of the Mcm2 interaction prevents DDK from efficiently stimulating the initiation of DNA replication. Following initiation, Mcm2-7 migrates with the replication fork. Interestingly, the motif C mutants were sensitive to long-term, but not short-term exposure to the genotoxic agents hydroxyurea and methyl methanesulfonate. These results support a model whereby DDK interaction with Mcm2 is important to stabilize and/or restart replication forks during conditions where a prolonged S-phase checkpoint is triggered.