Biochemical analysis of human PIF1 helicase and functions of its N-terminal domain.
Biochemical analysis of human PIF1 helicase and functions of its N-terminal domain.
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人PIF1解旋酶及其N末端结构域的功能的生化分析。
DOI：
10.1093/nar/gkn609
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发表时间：
2008-11
影响因子：
14.9
通讯作者：
Kamiya, Kenji
中科院分区：
文献类型：
--
作者：
Gu, Yongqing;Masuda, Yuji;Kamiya, Kenji
进化保守的PIF1 DNA解旋酶家族似乎在很大程度上具有不重叠的细胞功能。为了更好地了解人PIF1的功能，我们研究了该蛋白的生化性质。对单链(Ss)DNA依赖的ATPase活性的分析表明，由于对PIF1的高亲和力，非结构ssDNA可以极大地刺激ATPase活性，尽管PIF1优先解开分叉的底物。这表明PIF1需要一个单链DNA区域来装载，一个叉形结构来进入双链区域。缺失分析显示了一个独特的N-末端部分的新功能，称为PIF1 N-末端(Pint)结构域。当pint结构域被截断时，即使ATPase活性的最大速度和对ATP的Km值没有影响，对ssDNA的表观亲和力和解离活性也大大降低。我们认为pint结构域通过其固有的结合活性有助于增强与单链DNA的相互作用。此外，我们还发现了DNA链退火活性，也位于品脱结构域。值得注意的是，PIF1的解离和退火活性被复制蛋白A抑制。这些结果表明，PIF1的功能可能受到特定情况和DNA结构的限制。
The evolutionary conserved PIF1 DNA helicase family appears to have largely nonoverlapping cellular functions. To better understand the functions of human PIF1, we investigated biochemical properties of this protein. Analysis of single-stranded (ss) DNA-dependent ATPase activity revealed nonstructural ssDNA to greatly stimulate ATPase activity due to a high affinity for PIF1, even though PIF1 preferentially unwinds forked substrates. This suggests that PIF1 needs a ssDNA region for loading and a forked structure for translocation entrance into a double strand region. Deletion analysis demonstrated novel functions of a unique N-terminal portion, named the PIF1 N-terminal (PINT) domain. When the PINT domain was truncated, apparent affinity for ssDNA and unwinding activity were much reduced, even though the maximum velocity of ATPase activity and Km value for ATP were not affected. We suggest that the PINT domain contributes to enhancing the interaction with ssDNA through intrinsic binding activity. In addition, we found DNA strand-annealing activity, also residing in the PINT domain. Notably, the unwinding and annealing activities were inhibited by replication protein A. These results suggest that the functions of PIF1 might be restricted with particular situations and DNA structures.
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通讯作者：
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