Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
J Biol Chem. 2012 Sep 21;287(39):32953-66. doi: 10.1074/jbc.M112.384321. Epub 2012 Jul 30.
The Escherichia coli very short patch (VSP) repair pathway corrects thymidine-guanine mismatches that result from spontaneous hydrolytic deamination damage of 5-methyl cytosine. The VSP repair pathway requires the Vsr endonuclease, DNA polymerase I, a DNA ligase, MutS, and MutL to function at peak efficiency. The biochemical roles of most of these proteins in the VSP repair pathway have been studied extensively. However, these proteins have not been studied together in the context of VSP repair in an in vitro system. Using purified components of the VSP repair system in a reconstitution reaction, we have begun to develop an understanding of the role played by each of these proteins in the VSP repair pathway and have gained insights into their interactions. In this report we demonstrate an in vitro reconstitution of the VSP repair pathway using a plasmid DNA substrate. Surprisingly, the repair track length can be modulated by the concentration of DNA ligase. We propose roles for MutL and MutS in coordination of this repair pathway.
大肠杆菌非常短补丁（VSP）修复途径纠正胸腺嘧啶 - 鸟嘌呤错配，导致自发水解脱氨损伤的 5-甲基胞嘧啶。VSP 修复途径需要 Vsr 内切酶，DNA 聚合酶 I，DNA 连接酶，MutS 和 MutL 以最高效率发挥作用。这些蛋白质在 VSP 修复途径中的大多数生化作用已得到广泛研究。然而，这些蛋白质尚未在体外系统中的 VSP 修复背景下一起进行研究。使用 VSP 修复系统的纯化成分在重建反应中，我们开始了解这些蛋白质在 VSP 修复途径中的作用，并深入了解它们的相互作用。在本报告中，我们使用质粒 DNA 底物证明了 VSP 修复途径的体外重建。令人惊讶的是，DNA 连接酶的浓度可以调节修复轨道的长度。我们提出了 MutL 和 MutS 在协调该修复途径中的作用。
