Robertson Adam, Pattishall Steven R, Matson Steven W
Department of Biology, Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
J Biol Chem. 2006 Mar 31;281(13):8399-408. doi: 10.1074/jbc.M509184200. Epub 2006 Jan 30.
The DNA binding properties of the mismatch repair protein MutL and their importance in the repair process have been controversial for nearly two decades. We have addressed this issue using a point mutant of MutL (MutL-R266E). The biochemical and genetic data suggest that DNA binding by MutL is required for dam methylation-directed mismatch repair. We demonstrate that purified MutL-R266E retains wild-type biochemical properties that do not depend on DNA binding, such as basal ATP hydrolysis in the absence of DNA and the ability to interact with other mismatch repair proteins. However, purified MutL-R266E binds DNA poorly in vitro as compared with MutL, and consistent with this observation, its DNA-dependent biochemical activities, like DNA-stimulated ATP hydrolysis and helicase II stimulation, are severely compromised. In addition, there is a modest effect on stimulation of MutH-catalyzed nicking. Finally, genetic assays show that MutL-R266E has a strong mutator phenotype, demonstrating that the mutant is unable to function in dam methylation-directed mismatch repair in vivo.
错配修复蛋白MutL的DNA结合特性及其在修复过程中的重要性在近二十年来一直存在争议。我们使用MutL的一个点突变体（MutL-R266E）解决了这个问题。生化和遗传数据表明，MutL与DNA的结合是dam甲基化导向的错配修复所必需的。我们证明，纯化的MutL-R266E保留了不依赖于DNA结合的野生型生化特性，如在无DNA时的基础ATP水解以及与其他错配修复蛋白相互作用的能力。然而，与MutL相比，纯化的MutL-R266E在体外与DNA的结合能力较差，与此观察结果一致，其依赖于DNA的生化活性，如DNA刺激的ATP水解和对解旋酶II的刺激，都受到严重损害。此外，对MutH催化的切口有适度的刺激作用。最后，遗传分析表明MutL-R266E具有强烈的突变表型，证明该突变体在体内无法在dam甲基化导向的错配修复中发挥作用。
