Program of Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, New York 10065.
Program of Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, New York 10065.
J Biol Chem. 2010 Mar 26;285(13):9493-9505. doi: 10.1074/jbc.M109.093609. Epub 2010 Jan 19.
Ctf4/AND-1 is a highly conserved gene product required for both DNA replication and the establishment of sister chromatid cohesion. In this report, we examined the mechanism of action of human Ctf4 (hCtf4) in DNA replication both in vitro and in vivo. Our findings show that the purified hCtf4 exists as a dimer and that the hCtf4 SepB domain likely plays a primary role determining the dimeric structure. hCtf4 binds preferentially to DNA template-primer structures, interacts directly with the replicative DNA polymerases (alpha, delta, and epsilon), and markedly stimulates the polymerase activities of DNA polymerases alpha and epsilon in vitro. Depletion of hCtf4 in HeLa cells by small interfering RNA resulted in G(1)/S phase arrest. DNA fiber analysis revealed that cells depleted of hCtf4 exhibited a rate of DNA replication slower than cells treated with control small interfering RNA. These findings suggest that in human cells, hCtf4 plays an essential role in DNA replication and its ability to stimulate the replicative DNA polymerases may contribute to this effect.
Ctf4/AND-1 是一种高度保守的基因产物，对于 DNA 复制和姐妹染色单体黏合的建立都是必需的。在本报告中，我们研究了人 Ctf4（hCtf4）在体外和体内的 DNA 复制中的作用机制。我们的发现表明，纯化的 hCtf4 以二聚体的形式存在，并且 hCtf4 的 SepB 结构域可能在决定二聚体结构方面起主要作用。hCtf4 优先结合 DNA 模板-引物结构，与复制 DNA 聚合酶（α、δ和ε）直接相互作用，并显著刺激体外 DNA 聚合酶α和ε的聚合酶活性。通过小干扰 RNA 耗尽 HeLa 细胞中的 hCtf4 导致 G1/S 期停滞。DNA 纤维分析表明，耗尽 hCtf4 的细胞的 DNA 复制速度比用对照小干扰 RNA 处理的细胞慢。这些发现表明，在人细胞中，hCtf4 在 DNA 复制中发挥着重要作用，其刺激复制 DNA 聚合酶的能力可能促成了这种作用。
