Studies with the human cohesin establishment factor, ChlR1 - Association of ChlR1 with Ctf18-RFC and Fen1
Studies with the human cohesin establishment factor, ChlR1 - Association of ChlR1 with Ctf18-RFC and Fen1
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DOI：
10.1074/jbc.m802696200
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发表时间：
2008-07-25
影响因子：
4.8
通讯作者：
Hurwitz, Jerard
中科院分区：
文献类型：
--
作者：
Farina, Andrea;Shin, Jae-Ho;Hurwitz, Jerard
人类ChlR1(HChlR1)是解旋酶DEAD/Deah亚家族的成员之一，被证明与粘附素复合体的组成部分相互作用，并在姐妹染色单体凝聚中发挥作用。为了研究hChlR1的生化和生物学特性，我们从293细胞中纯化了hChlR1蛋白，证明hChlR1具有DNA依赖的ATPase和解旋酶活性。这种解旋酶在有ATP和少量dATP存在的情况下，以5‘到3’的方向在单链DNA上移位。它的解离活性需要5‘-单链区域来装载解旋酶，因为平端的双链结构不支持解离。HChlR1的解旋酶活性能够取代长达100bp的双链区，这一双链区可以被RPA或凝聚力建立因子Ctf18-RFC(复制因子C)复合体延伸到500bp。我们发现hChlR1与hCtf18-RFC复合体、人增殖细胞核抗原和hFen1相互作用。Fen1和hChlR1之间的相互作用刺激了Fen1的翻盖内切酶活性。通过靶向的小干扰RNA处理选择性地耗尽hChlR1或Fen1会导致姐妹染色单体的早熟分离。这些发现与hChlR1在姐妹染色单体凝聚力建立中的作用是一致的，并表明它的作用可能有助于滞后的链加工事件在凝聚力中起重要作用。
Human ChlR1 (hChlR1), a member of the DEAD/DEAH subfamily of helicases, was shown to interact with components of the cohesin complex and play a role in sister chromatid cohesion. In order to study the biochemical and biological properties of hChlR1, we purified the protein from 293 cells and demonstrated that hChlR1 possesses DNA-dependent ATPase and helicase activities. This helicase translocates on single-stranded DNA in the 5' to 3' direction in the presence of ATP and, to a lesser extent, dATP. Its unwinding activity requires a 5'-single-stranded region for helicase loading, since flush-ended duplex structures do not support unwinding. The helicase activity of hChlR1 is capable of displacing duplex regions up to 100 bp, which can be extended to 500 bp by RPA or the cohesion establishment factor, the Ctf18-RFC (replication factor C) complex. We show that hChlR1 interacts with the hCtf18-RFC complex, human proliferating cell nuclear antigen, and hFen1. The interactions between Fen1 and hChlR1 stimulate the flap endonuclease activity of Fen1. Selective depletion of either hChlR1 or Fen1 by targeted small interfering RNA treatment results in the precocious separation of sister chromatids. These findings are consistent with a role of hChlR1 in the establishment of sister chromatid cohesion and suggest that its action may contribute to lagging strand processing events important in cohesion.