Mismatch Repair in Gamma-Proteobacteria

γ-变形杆菌的错配修复 项目摘要/摘要 错配修复(MMR)是一种高度保守的基因组维护过程，主要解决 聚合酶错误结合错误。MMR基因突变显著增加自发性 突变率，并与适应以及相关的多药耐药有关 病原菌。人类MMR突变是常见人类癌症的原因 易感林奇综合征以及许多散发性癌症。 MMR是在DNA链断裂时启动的切除-再合成过程，可能有数百条DNA链断裂 远离错配的数千个核苷酸。MMR的保真度取决于建立 单链DNA在含有错误的DNA链上断裂。变态γ细菌的一个子集，包括E.Coli和 ESKAPE病原体克雷伯氏菌和肠杆菌，最近进化的DNA腺嘌呤甲基化(DAM)和 MUT将单链DNA断裂引入到新复制的包含误结合错误的链上。 利用Dam/Muth MMR途径的细菌与所有其他细菌之间的一个重要区别 生物体中的DAM突变与几个同源基因的突变结合在一起是致命的 重组基因(合成致命性)。同样，MMR切除成分的突变在 结合复制编辑基因突变。导致MMR依赖的机制 合成杀伤力在很大程度上是假想的。 确定性模型历来支持MMR机制，其中确定的络合物和 提出了完成生化事件的进展。使用新开发的单分子- 我们的成像技术表明，MMR的初始步骤依赖于随机DNA扩散机制 它们是由陆地生物学中两个最保守的MMR蛋白MutS和MutL调控的。 这一新的应用程序将测试整个多组分MMR切除过程是 随机性(完全由随机过程支配；与确定性完全相反)。理解 这种生化随机性应该会影响针对MMR的未来研究和治疗策略。 我们建议使用体外和体内的实时单分子成像来解决 E.ColiMMR及其在合成致死中的作用。具体目标是：1.定量生物物理成像 单个MMR组件活动，2.)MMR完全切除反应的可视化 单个不匹配的DNA分子，以及3.)活细胞中MMR组分相互作用的分析。

MISMATCH REPAIR IN γ-PROTEOBACTERIA PROJECT SUMMARY / ABSTRACT Mismatch repair (MMR) is a highly conserved genome maintenance process that primarily resolves polymerase misincorporation errors. Mutation of the MMR genes dramatically increase spontaneous mutation rates and have been linked to adaptation as well as associated multi-drug resistance in several pathogenic bacteria. MMR mutations in humans are the cause of the common human cancer predisposition Lynch syndrome as well as numerous sporadic cancers. MMR is an excision-resynthesis process that is initiated at a DNA strand break, which may be hundreds to thousands of nucleotides away from the mismatch. The fidelity of MMR depends on establishing the ssDNA break on the error-containing DNA strand. A subset of γ-proteobacteria, including E.coli and the ESKAPE pathogens Klebsiella and Enterobacter, recently evolved DNA adenine methylation (Dam) and MutH to introduce a ssDNA break onto the newly replicated strand containing a misincorporation error. An important distinction between bacteria that utilize the Dam/MutH MMR Pathway and all other organisms is that dam mutations are lethal in combination with mutation of several homologous recombination genes (synthetic lethality). Similarly, mutations of MMR excision components are lethal in combination with replication editing gene mutations. The mechanisms that lead to MMR-dependent synthetic lethality are largely hypothetical. Deterministic models have historically underpinned MMR mechanisms, where definite complexes and progressions are proposed to complete the biochemical events. Using newly developed single molecule- imaging techniques we showed that the initial steps of MMR rely on random DNA diffusion mechanics that are modulated by the two most highly conserved MMR proteins in terrestrial biology, MutS and MutL. This new application will test the hypothesis that the entire multi-component MMR excision process is Stochastic (fully governed by random processes; the complete opposite of Deterministic). Understanding such biochemical randomness should impact future research and therapeutic strategies targeting MMR. We propose to use real-time single molecule imaging in vitro and in vivo to resolve the mechanics of E.coli MMR and its role in synthetic lethality. The Specific Aims are: 1.) quantitative biophysical imaging of individual MMR component activities, 2.) visualization of the complete MMR excision reaction on single mismatched DNA molecules, and 3.) analysis of MMR component interactions in live cells.