Recognition of forked and single-stranded DNA structures by human RAD18 complexed with RAD6B protein triggers its recruitment to stalled replication forks
Recognition of forked and single-stranded DNA structures by human RAD18 complexed with RAD6B protein triggers its recruitment to stalled replication forks
复制标题
DOI：
10.1111/j.1365-2443.2008.01176.x
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发表时间：
2008-04-01
期刊：
影响因子：
2.1
通讯作者：
Tateishi, Satoshi
中科院分区：
文献类型：
--
作者：
Tsuji, Yuri;Watanabe, Kenji;Tateishi, Satoshi
复制后的DNA修复有助于在DNA损伤位点的复制叉停止时恢复DNA合成。尽管RAD18和聚合酶（Pol eta）对复制后修复（PRR）很重要，但这些因子被招募来阻止复制分叉的分子机制尚不清楚。我们提供的证据表明，人类RAD18与RAD6B蛋白复合物优先结合到分叉和单链DNA （ssDNA）结构上，这些结构已知位于停滞的复制分叉处。RAD18的SAP结构域（残基248-282）是RAD18与RAD6B复合物与DNA底物结合的关键。在体内，SAP结构域突变的RAD18不能在DNA损伤位点积累，也不能引导DNA Pol eta停止复制分叉。SAP结构域也是高效的PCNA单泛素化所必需的。SAP结构域突变体不能抑制rad18敲除细胞的紫外线敏感性。这些结果表明，RAD18与RAD6B复合物通过与分叉DNA或长ssDNA结构的相互作用来阻止复制分叉，这是启动PRR所需的一个过程。
Post-replication DNA repair facilitates the resumption of DNA synthesis upon replication fork stalling at DNA damage sites. Despite the importance of RAD18 and polymerase eta (Pol eta) for post-replication repair (PRR), the molecular mechanisms by which these factors are recruited to stalled replication forks are not well understood. We present evidence that human RAD18 complexed with RAD6B protein preferentially binds to forked and single-stranded DNA (ssDNA) structures, which are known to be localized at stalled replication forks. The SAP domain of RAD18 (residues 248-282) is crucial for binding of RAD18 complexed with RAD6B to DNA substrates. RAD18 mutated in the SAP domain fails to accumulate at DNA damage sites in vivo and does not guide DNA Pol eta to stalled replication forks. The SAP domain is also required for the efficient mono-ubiquitination of PCNA. The SAP domain mutant fails to suppress the ultraviolet (UV)-sensitivity of Rad18-knockout cells. These results suggest that RAD18 complexed with RAD6B is recruited to stalled replication forks via interactions with forked DNA or long ssDNA structures, a process that is required for initiating PRR.