Department of Biochemistry and Howard Hughes Medical Institute, Box 3711, Duke University Medical Center, Durham, NC 27710.
Proc Natl Acad Sci U S A. 2010 Sep 14;107(37):16066-71. doi: 10.1073/pnas.1010662107. Epub 2010 Aug 16.
MutLα (MLH1-PMS2) is a latent endonuclease that is activated in a mismatch-, MutSα-, 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 MutLα activation, but in this case cleavage strand bias is largely abolished. Based on these findings we suggest that PCNA has two roles in MutLα 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 MutLα 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.
MutLα（MLH1-PMS2）是一种潜伏的内切核酸酶，以错配、MutSα、增殖细胞核抗原（PCNA）、复制因子 C（RFC）和 ATP 依赖性方式激活，其核酸酶活性定向于含有预先存在断裂的异源双链链。RFC 耗竭实验和线性 DNA 的使用表明，RFC 在内切核酸酶激活中的功能仅限于 PCNA 加载。虽然带有切口的环形异源双链 DNA 是 PCNA 加载和切口链内切核酸酶激活的良好底物，但共价闭合、松弛的环形 DNA 是这两种反应的不良底物。然而，共价闭合的超螺旋或含有泡的松弛异源双链体，虽然支持 PCNA 加载，但也支持 MutLα 激活，但在这种情况下，切割链偏向被大大消除。基于这些发现，我们认为 PCNA 在 MutLα 功能中具有两个作用：夹钳是内切核酸酶激活所必需的，这种效应显然涉及两种蛋白质的相互作用，并且由于其加载取向，PCNA 决定了 MutLα 切口的链方向。这些结果还为非复制 DNA 上错配修复的激活提供了一种潜在的机制，这可能对三核苷酸重复扩展的体细胞阶段具有影响。
