Budding Yeast Dbf4 Sequences Required for Cdc7 Kinase Activation and Identification of a Functional Relationship Between the Dbf4 and Rev1 BRCT Domains
Budding Yeast Dbf4 Sequences Required for Cdc7 Kinase Activation and Identification of a Functional Relationship Between the Dbf4 and Rev1 BRCT Domains
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DOI：
10.1534/genetics.109.110155
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发表时间：
2009-12-01
期刊：
影响因子：
3.3
通讯作者：
Weinreich, Michael
中科院分区：
文献类型：
--
作者：
Harkins, Victoria;Gabrielse, Carrie;Weinreich, Michael
Cdc 7-Dbf 4是启动DNA复制所需的双亚基激酶。Dbf 4调节亚基是Cdc 7激酶活性所必需的。先前的研究已经表明Dbf 4直系同源物的C末端编码单个（推定的）C2 H2锌（Zn）指，称为“基序C”。“通过突变分析，我们表明锌指不是。这是Dbf 4的基本功能所必需的。然而，删除和点突变改变保守的锌指残基表现出显着放缓的S-期，DNA损伤的敏感性，和低诱变表型紫外线照射后。使用双杂交和生化分析，我们表明，Dbf 4锌指与Cdc 7相互作用，并刺激其激酶活性。然而，一个可分离的Dbf 4区域也介导与Cdc 7的相互作用，因此只有两个Cdc 7相互作用区域的缺失才导致致死性。相反，N-末端BRCT样结构域不是。也不与Cdc 7相互作用。通过在酿酒酵母中制备含有已知BRCT结构域的嵌合Dbf 4蛋白，我们表明BRCT结构域前端Rev 1，一种跨损伤DNA聚合酶，可以独特地取代Dbf 4 BRCT结构域。因此，我们已经绘制了芽殖酵母Dbf 4上结合和激活Cdc 7激酶所需的区域。我们的数据还表明，Dbf 4和Rev 1 BRCT结构域与一个共同的蛋白质或结构相互作用，尽管这两个结构域及其结合伙伴的精确功能仍然难以捉摸。
Cdc7-Dbf4 is a two-subunit kinase required for initiating DNA replication. The Dbf4 regulatory subunit is required for Cdc7 kinase activity. Previous studies have shown that the C termini of Dbf4 orthologs encode a single (putative) C2H2 zinc (Zn) finger, referred to as "motif C." By mutational analysis we show that the Zn finger is not. required for the essential function of Dbf4. However, deletion and point mutants altering conserved Zn-finger residues exhibit a substantially slowed S-phase, DNA damage sensitivity, and a hypo-mutagenic phenotype following UV irradiation. Using two-hybrid and biochemical assays, we show that the Dbf4 Zn finger interacts with Cdc7 and stimulates its kinase activity. However, a separable Dbf4 region also mediates an interaction with Cdc7 Such that only the loss of both Cdc7-interacting regions results in lethality. In contrast, an N-terminal BRCT-like domain is not. required for induced mutagenesis nor does it interact with Cdc7. By making chimeric Dbf4 proteins that contain known BRCT domains in Saccharomyces cerevisiae, We show that the BRCT domain front Rev1, a translesion DNA polymerase, can uniquely substitute for the Dbf4 BRCT domain. Thus, We have mapped regions on budding yeast Dbf4 required for binding and activating Cdc7 kinase. Our data also Suggest that the Dbf4 and Rev1 BRCT domains interact with a common protein or structure, although the precise function of both domains and their binding Partners remains elusive.