PCNA function in the activation and strand direction of MutLα endonuclease in mismatch repair
PCNA function in the activation and strand direction of MutLα endonuclease in mismatch repair
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DOI：
10.1073/pnas.1010662107
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发表时间：
2010-09-14
影响因子：
11.1
通讯作者：
Modrich, Paul
中科院分区：
文献类型：
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作者：
Pluciennik, Anna;Dzantiev, Leonid;Modrich, Paul
MutL α (MLH1-PMS2)是一种潜在的内切酶，它以错配、MutS α -、增殖细胞核抗原(PCNA)-、复制因子C (RFC)-和atp依赖的方式激活，核酸酶的作用指向含有预先存在断裂的异双链。RFC耗尽实验和线性dna的使用表明，RFC在内切酶激活中的功能仅限于PCNA装载。然而，有缺口的环状异双工DNA是PCNA装载和在切割链上激活内切酶的良好底物，而共价闭合的松弛环状DNA对于这两种反应都是较差的底物。然而，共价闭合的超螺旋或含气泡的松弛异双工，支持PCNA装载，也支持MutLa激活，但在这种情况下，切割链的偏置很大程度上被消除了。基于这些发现，我们认为PCNA在MutLa功能中有两个作用：夹子是核酸内切酶激活所必需的，这一作用显然涉及两种蛋白质的相互作用，并且PCNA通过其装载方向决定MutLa切口的链方向。这些结果也为非复制DNA错配修复的激活提供了一种潜在的机制，这种效应可能对三联体重复扩增的体细胞阶段有影响。
MutL alpha (MLH1-PMS2) is a latent endonuclease that is activated in a mismatch-, MutS alpha-, proliferating cell nuclear antigen (PCNA)-, replication factor C (RFC)-, and ATP-dependent manner, with nuclease action directed to the heteroduplex strand that contains a preexisting break. RFC depletion experiments and use of linear DNAs indicate that RFC function in endonuclease activation is limited to PCNA loading. Whereas nicked circular heteroduplex DNA is a good substrate for PCNA loading and for endonuclease activation on the incised strand, covalently closed, relaxed circular DNA is a poor substrate for both reactions. However, covalently closed supercoiled or bubble-containing relaxed heteroduplexes, which do support PCNA loading, also support MutLa activation, but in this case cleavage strand bias is largely abolished. Based on these findings we suggest that PCNA has two roles in MutLa function: The clamp is required for endonuclease activation, an effect that apparently involves interaction of the two proteins, and by virtue of its loading orientation, PCNA determines the strand direction of MutLa incision. These results also provide a potential mechanism for activation of mismatch repair on nonreplicating DNA, an effect that may have implications for the somatic phase of triplet repeat expansion.