Fukui Kenji, Nishida Masami, Nakagawa Noriko, Masui Ryoji, Kuramitsu Seiki
RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.
J Biol Chem. 2008 May 2;283(18):12136-45. doi: 10.1074/jbc.M800110200. Epub 2008 Feb 29.
DNA mismatch repair corrects mismatched base pairs mainly caused by replication error. Recent studies revealed that human MutL endonuclease, hPMS2, plays an essential role in the repair. However, there has been little biochemical analysis of the MutL endonuclease. In particular, it is unknown for what the MutL utilizes ATP binding and hydrolyzing activity. Here we report the detailed functional analysis of Thermus thermophilus MutL (ttMutL). ttMutL exhibited an endonuclease activity that decreased on alteration of Asp-364 in ttMutL to Asn. The biochemical characteristics of ttMutL were significantly affected on ATP binding, which suppressed nonspecific DNA digestion and promoted the mismatch- and MutS-dependent DNA binding. The inactivation of the cysteinyl residues in the C-terminal domain resulted in the perturbation in ATP-dependent regulation of the endonuclease activity, although the ATP-binding motif is located in the N-terminal domain. Complementation experiments revealed that the endonuclease activity of ttMutL and its regulation by ATP binding are necessary for DNA repair in vivo.
DNA错配修复主要纠正由复制错误导致的错配碱基对。最近的研究表明，人类MutL核酸内切酶hPMS2在修复过程中起着至关重要的作用。然而，对MutL核酸内切酶的生化分析却很少。特别是，尚不清楚MutL利用ATP结合和水解活性的目的。在此，我们报告嗜热栖热菌MutL（ttMutL）的详细功能分析。ttMutL表现出一种核酸内切酶活性，当ttMutL中的天冬氨酸-364被替换为天冬酰胺时，该活性降低。ttMutL的生化特性在ATP结合时受到显著影响，这抑制了非特异性DNA消化并促进了错配和MutS依赖性DNA结合。尽管ATP结合基序位于N端结构域，但C端结构域中半胱氨酸残基的失活导致了核酸内切酶活性的ATP依赖性调节受到干扰。互补实验表明，ttMutL的核酸内切酶活性及其通过ATP结合的调节对于体内DNA修复是必需的。
