Enzymology Of Eukaryotic Mismatch Repair

 描述(申请人提供)：DNA错配修复是突变产生的主要调节因素。该途径具有多种避免突变的功能，包括纠正DNA生物合成错误，抑制准同源序列之间的重组，以及参与细胞对DNA损伤的反应。人类错配修复的失活是最常见的遗传性癌症林奇综合征的原因。错配修复缺陷也对癌症治疗有影响，因为该途径的失活使细胞对某些抗肿瘤药物的细胞毒作用产生抗药性，这是其在DNA损伤反应中的功能的结果。令人惊讶的是，错配修复系统的组件也是产生某些突变所必需的，包括扩展的三联体重复序列，这是许多神经退行性疾病的原因。我们感兴趣的是错配修复功能在控制突变产生中的分子机制。为此，我们建议继续进行四项工作：(1)MutLα与MutSα的相互作用被认为在错配修复的启动中发挥重要作用，而增殖细胞核抗原复制钳与MutLα的相互作用是激活MutLα核酸内切酶和链方向所必需的。我们将建立这些络合物的分子化学计量学，并进一步澄清它们的性质。在增殖细胞核抗原*MutLα复合体的情况下，我们还将试图确定相互作用所需的MutLα基序(S)。该项目的这一方面将包括对锌在Mut�内切酶功能中的作用进行评估。(2)(Cag)n/(Ctg)n神经退行性疾病的体细胞扩张期可发生在有丝分裂后细胞中，这取决于错配修复活性MutSβ、MutLα和MutLγ。我们已经发现，在共价连续的DNA中，小的CAG或CTG挤出可以作为非规范的增殖细胞核抗原装载位点，从而导致MutSβ依赖的MutLα内切酶的激活，这为非复制DNA的修复激活提供了一种简单的机制。我们正在将这些研究扩展到分析在提取和纯化系统中人类错配修复系统对(CAG)n/(CTG)n重复处理的分析。平行研究将解决扩展的(GaA)n/(Ttc)n Friedreich‘s共济失调等位基因的不稳定性，这些等位基因以MutSβ−和MutLα内切酶依赖的方式受到双链切割。(3)错配修复功能是检验点和对O6-甲基鸟嘌呤的凋亡反应所必需的，O6-甲基鸟嘌呤是SN1 DNA甲基化子产生的细胞毒性损伤。模板链上含有O6-甲基鸟嘌呤的异源双链DNA支持流产切除和修复的迭代循环，该循环在细胞提取液中持续数小时。我们将试图澄清这种反应的性质。(4)与洛雷纳·比斯实验室正在进行的合作研究将探讨人类错配修复系统处理脱氧核糖核酸的结构特征，重点是增殖细胞核抗原*MutLα复合体。

 DESCRIPTION (provided by applicant): DNA mismatch repair is a primary regulator of mutation production. The pathway is well known for its multiple mutation-avoidance functions, including the correction of DNA biosynthetic errors, inhibition of recombination between quasi-homologous sequences, and participation in the cellular response to DNA damage. Inactivation of human mismatch repair is the cause of Lynch syndrome, the most common hereditary cancer. Mismatch repair defects also have implications for cancer treatment because inactivation of the pathway renders cells resistant to the cytotoxic effects of certain anti-tumor drugs, a consequence of its function in the DNA damage response. Surprisingly, components of the mismatch repair system are also required for the generation of certain mutations including expanded triplet repeat sequences which are the cause of a number of neurodegenerative diseases. We are interested in the molecular mechanisms responsible for mismatch repair function in the control of mutation production. To this end we propose continuation of four ongoing lines of work: (1) MutLα interaction with MutSα is believed to play an important role in the initiation of mismatch repair, while interaction of the PCNA replication clamp with MutLα is required for activation and strand direction of the MutLα endonuclease. We will establish the molecular stoichiometries of these complexes and pursue further clarification of their nature. In the case of the PCNA*MutLα complex, we will also attempt to identify MutLα motif(s) required for the interaction. This aspect of the project will include evaluation of the role of zinc in Mut� endonuclease function. (2) The somatic expansion stage of (CAG)n/(CTG)n neurodegenerative diseases, which depends on the mismatch repair activities MutSβ, MutLα and MutLγ, can occur in post-mitotic cells. We have found that small CAG or CTG extrusions in covalently continuous DNA can serve as noncanonical PCNA loading sites, thus resulting in MutSβ-dependent activation of MutLα endonuclease, which provides a simple mechanism for repair activation on non-replicating DNA. We are extending these studies to the analysis of (CAG)n/(CTG)n repeat processing by the human mismatch repair system in both extract and purified systems. Parallel studies will address the instability of expanded (GAA)n/(TTC)n Friedreich's ataxia disease alleles, which are subject to double strand cleavage in a MutSβ− and MutLα endonuclease-dependent manner. (3) Mismatch repair function is required for checkpoint and apoptotic responses to O6-methylguanine, the cytotoxic lesion produced by SN1 DNA methylators. Heteroduplex DNA that contains O6-methylguanine on the template strand supports iterative cycles of abortive excision and repair that continues for several hours in cell extracts. We will attempt to clarify the nature of this reaction. (4) Ongoing collaborative studies with the laboratoy of Lorena Beese will address structural features of DNA processing by the human mismatch repair system with emphasis on the PCNA*MutLα complex.