Cross talk between the nuclease and helicase activities of Dna2: role of an essential iron-sulfur cluster domain.
Cross talk between the nuclease and helicase activities of Dna2: role of an essential iron-sulfur cluster domain.
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DOI：
10.1093/nar/gks534
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发表时间：
2012-09
影响因子：
14.9
通讯作者：
Campbell JL
中科院分区：
文献类型：
--
作者：
Pokharel S;Campbell JL
DNA 2核酸酶/解旋酶是参与DNA复制和重组修复的多任务蛋白，对于保持基因组稳定性非常重要。酵母Dna 2蛋白含有一个保守的推定的Fe-S（铁-硫）簇签名基序跨越核酸酶活性位点。我们表明，这个基序确实是一个Fe-S簇结构域。参与金属配位的半胱氨酸的突变不仅大大降低了核酸酶活性，而且还降低了DNA 2的ATP酶活性，这表明核酸酶和解旋酶活性是偶联的。对DNA的亲和力没有显著降低，但C至A突变体中的结合模式改变。值得注意的是，接近Fe-S簇结构域的点突变（P504 S）使细胞对温度敏感，密切模仿Fe-S簇突变本身的全局缺陷。这表明了这种保守的脯氨酸残基在稳定Fe-S簇中的重要作用。C至A突变体在体内DNA复制和修复中有缺陷，并且引人注目的是，它们的缺陷程度与酶活性的丧失程度直接相关。与以前的结果表明，在ATP结构域的突变影响核酸酶的功能，我们的研究结果提供了一个新的机制之间的耦合在同一多肽中融合的核酸酶和解旋酶模块的范例。
Dna2 nuclease/helicase is a multitasking protein involved in DNA replication and recombinational repair, and it is important for preservation of genomic stability. Yeast Dna2 protein contains a conserved putative Fe–S (iron–sulfur) cluster signature motif spanning the nuclease active site. We show that this motif is indeed an Fe–S cluster domain. Mutation of cysteines involved in metal coordination greatly reduces not just the nuclease activity but also the ATPase activity of Dna2, suggesting that the nuclease and helicase activities are coupled. The affinity for DNA is not significantly reduced, but binding mode in the C to A mutants is altered. Remarkably, a point mutation (P504S), proximal to the Fe–S cluster domain, which renders cells temperature sensitive, closely mimics the global defects of the Fe–S cluster mutation itself. This points to an important role of this conserved proline residue in stabilizing the Fe–S cluster. The C to A mutants are deficient in DNA replication and repair in vivo, and, strikingly, the degree to which they are defective correlates directly with degree of loss of enzymatic activity. Taken together with previous results showing that mutations in the ATP domain affect nuclease function, our results provide a new mechanistic paradigm for coupling between nuclease and helicase modules fused in the same polypeptide.
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影响因子：
3.7
作者：
Budd ME;Campbell JL
通讯作者：
Campbell JL
影响因子：
4.5
作者：
Budd ME;Tong AH;Polaczek P;Peng X;Boone C;Campbell JL
通讯作者：
Campbell JL
影响因子：
14.9
作者：
Bae, SH;Kim, JA;Seo, YS
通讯作者：
Seo, YS
DOI：
10.1073/pnas.0906429106
发表时间：
2009-08-11
影响因子：
11.1
作者：
Lee, Paul E.;Demple, Bruce;Barton, Jacqueline K.
通讯作者：
Barton, Jacqueline K.
影响因子：
4.8
作者：
Bae, SH;Kim, DW;Seo, YS
通讯作者：
Seo, YS