Heinze Roger J, Giron-Monzon Luis, Solovyova Alexandra, Elliot Sarah L, Geisler Sven, Cupples Claire G, Connolly Bernard A, Friedhoff Peter
Institut für Biochemie, Justus-Liebig-Universität, D-35392 Giessen, Germany.
Nucleic Acids Res. 2009 Jul;37(13):4453-63. doi: 10.1093/nar/gkp380. Epub 2009 May 27.
DNA mismatch repair (MMR) and very-short patch (VSP) repair are two pathways involved in the repair of T:G mismatches. To learn about competition and cooperation between these two repair pathways, we analyzed the physical and functional interaction between MutL and Vsr using biophysical and biochemical methods. Analytical ultracentrifugation reveals a nucleotide-dependent interaction between Vsr and the N-terminal domain of MutL. Using chemical crosslinking, we mapped the interaction site of MutL for Vsr to a region between the N-terminal domains similar to that described before for the interaction between MutL and the strand discrimination endonuclease MutH of the MMR system. Competition between MutH and Vsr for binding to MutL resulted in inhibition of the mismatch-provoked MutS- and MutL-dependent activation of MutH, which explains the mutagenic effect of Vsr overexpression. Cooperation between MMR and VSP repair was demonstrated by the stimulation of the Vsr endonuclease in a MutS-, MutL- and ATP-hydrolysis-dependent manner, in agreement with the enhancement of VSP repair by MutS and MutL in vivo. These data suggest a mobile MutS-MutL complex in MMR signalling, that leaves the DNA mismatch prior to, or at the time of, activation of downstream effector molecules such as Vsr or MutH.
DNA错配修复（MMR）和极短补丁（VSP）修复是参与T:G错配修复的两条途径。为了解这两条修复途径之间的竞争与合作关系，我们使用生物物理和生化方法分析了MutL和Vsr之间的物理及功能相互作用。分析超速离心显示Vsr与MutL的N端结构域之间存在核苷酸依赖性相互作用。通过化学交联，我们将MutL与Vsr的相互作用位点定位到N端结构域之间的一个区域，该区域与之前描述的MutL与MMR系统的链区分内切酶MutH之间的相互作用区域相似。MutH和Vsr竞争与MutL结合导致错配引发的MutS和MutL依赖性MutH激活受到抑制，这解释了Vsr过表达的诱变作用。MMR和VSP修复之间的合作通过MutS、MutL和ATP水解依赖性方式刺激Vsr内切酶得以证明，这与体内MutS和MutL增强VSP修复的情况一致。这些数据表明在MMR信号传导中存在一个可移动的MutS-MutL复合物，该复合物在激活下游效应分子（如Vsr或MutH）之前或之时离开DNA错配位点。
