Fission Yeast Swi1-Swi3 Complex Facilitates DNA Binding of Mrc1*
Fission Yeast Swi1-Swi3 Complex Facilitates DNA Binding of Mrc1*
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DOI：
10.1074/jbc.m110.173344
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发表时间：
2010-10
期刊：
影响因子：
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通讯作者：
Taku Tanaka;M. Yokoyama;S. Matsumoto;Rino Fukatsu;Z. You;H. Masai
中科院分区：
文献类型：
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作者：
Taku Tanaka;M. Yokoyama;S. Matsumoto;Rino Fukatsu;Z. You;H. Masai
在裂殖酵母裂殖酵母的 DNA 复制过程中，复制叉保护复合体 Swi1-Swi3 和复制检查点介体 Mrc1 是维持复制叉完整性所必需的。这些蛋白质在稳定停滞的分叉和激活复制检查点信号通路方面发挥着至关重要的作用。尽管它们是保守的复制叉成分，但这些蛋白质的精确生化作用尚不清楚。在这里，我们纯化了 Mrc1 和 Swi1-Swi3 蛋白，并表明这些蛋白在体外独立但协同地与 DNA 结合。 Mrc1 优先结合停滞叉或 D 环样结构，尽管亲和力相对较低，而 Swi1-Swi3 复合物以更高的亲和力结合双链 DNA。在低浓度的 Swi1-Swi3 存在下，Mrc1 生成新型三元复合物，并以更高的亲和力与各种类型的 DNA 结合。此外，纯化的 Mrc1 和 Swi1-Swi3 在物理上彼此相互作用，并且这种相互作用因 Mrc1 的已知 DNA 结合域 (K235E、K236E) 的突变而消失。这种相互作用在 Swi1 (E662K) 的突变体中也消失了，这种突变体在称为 RTS1 的位点上的极性叉阻滞中特别有缺陷，并导致对基因毒性剂的敏感性，尽管 Swi1-Swi3 的 DNA 结合亲和力不受这种突变的影响。正如预期的那样，Swi1-Swi3 对 Mrc1 DNA 结合的协同效应也会因影响 Mrc1 和 Swi1-Swi3 之间相互作用的这些突变而丧失。我们的结果揭示了分子相互作用的一个方面，可能在复制暂停和叉稳定中发挥重要作用。
Replication fork protection complex Swi1-Swi3 and replication checkpoint mediator Mrc1 are required for maintenance of replication fork integrity during the course of DNA replication in the fission yeast Schizosaccharomyces pombe. These proteins play crucial roles in stabilizing stalled forks and activating replication checkpoint signaling pathways. Although they are conserved replication fork components, precise biochemical roles of these proteins are not known. Here we purified Mrc1 and Swi1-Swi3 proteins and show that these proteins bind to DNA independently but synergistically in vitro. Mrc1 binds preferentially to arrested fork or D-loop-like structures, although the affinity is relatively low, whereas the Swi1-Swi3 complex binds to double-stranded DNA with higher affinity. In the presence of a low concentration of Swi1-Swi3, Mrc1 generates a novel ternary complex and binds to various types of DNA with higher affinity. Moreover, purified Mrc1 and Swi1-Swi3 physically interact with each other, and this interaction is lost by mutations in the known DNA binding domain of Mrc1 (K235E,K236E). The interaction is also lost in a mutant form of Swi1 (E662K) that is specifically defective in polar fork arrest at a site called RTS1 and causes sensitivity to genotoxic agents, although the DNA binding affinity of Swi1-Swi3 is not affected by this mutation. As expected, the synergistic effect of the Swi1-Swi3 on DNA binding of Mrc1 is also lost by these mutations affecting the interaction between Mrc1 and Swi1-Swi3. Our results reveal an aspect of molecular interactions that may play an important role in replication pausing and fork stabilization.