hMLHI-hMRE11 interplay in DNA mismatch repair

描述（由申请方提供）：通过主要参与细胞周期调控、DNA复制、DNA修复和染色体分离的蛋白质网络，对从亲本细胞传递到后代的遗传信息的完整性进行细胞监测。该网络中任何蛋白质因子的缺陷都会使细胞在不同程度上遗传不稳定，因此易患癌症。DNA错配修复（MMR）系统是其中一个重要的途径，它通过监测和纠正DNA重组和DNA合成过程中发生的错配核苷酸，在维持基因组稳定性方面发挥着至关重要的作用。MMR基因的突变可导致更高的突变率并增加微卫星不稳定性。包括MutS和MutL同源物在内的几种MMR基因的种系突变有助于人类遗传性非息肉病性结直肠癌（HNPCC）的发病机制，其中hMLH 1和hMSH 2基因的突变是HNPCC的主要贡献者。我们最近的工作揭示了hMLH 1和hMRE 11之间的直接物理联系，在体外和体内。人hMRE 11代表多功能蛋白复合物（hRAD 50-hMRE 11-NBS 1）的必需核酸酶，其促进DNA双链断裂的修复，并在DNA损伤反应的信号传导以及减数分裂重组和端粒维持中起作用。hMRE 11的突变有助于罕见的“AT样”（ATLD）疾病的发展。已知hMRE 11相关复合物显示动态的空间和时间细胞分布，并在DNA损伤位点形成DNA损伤诱导的病灶。因此，该研究计划的长期目标是阐明MMR中涉及的分子机制和MMR蛋白与hMRE 11相关蛋白复合物之间的功能联系，以及破坏这些蛋白因子之间相互作用的突变的生物学效应。本研究拟通过系统的研究方法来解决这些重要问题，具体目标包括：（1）验证hMRE 11是DNA错配修复途径的功能组分的假设;（2）确定hMLH 1结合是否刺激hMRE 11的核酸酶活性以及hMRE 11的核酸酶活性在MMR过程中的功能作用;（3）检验hMLH 1 HNPCC错义突变对hMLH 1-hMRE 11和hMLH 1-hPMS 2杂合物形成的影响不同的假设，因此hMLH 1-hMRE 11复合物的破坏代表了hMLH 1 HNPCC突变致病作用的另一种机制。拟议研究的结果将提供深入了解MMR和hMRE 11相关复合物共同作用以维持遗传稳定性的分子机制，以及提供对人类疾病如癌症中hMLH 1和/或hMRE 11缺陷的更好理解。

DESCRIPTION (provided by applicant): Cellular surveillance for the integrity of genetic information passed from parental cells to subsequent generations is carried out by a network of proteins primarily involved in cell-cycle regulation, DNA replication, DNA repair, and chromosome segregation. Defects of any protein factor in this network render cells to genetic instability to different extents, and therefore, predispose to cancer. One of the involved pathways is the DNA mismatch repair (MMR) system that has a crucial role in maintaining genome stability by monitoring and correcting mismatched nucleotides occurred during DNA recombination and DNA synthesis. Mutations in MMR genes can cause higher mutation rates and increase microsatellite instability. Germline mutations in several MMR genes, including MutS and MutL homologues, contribute to the pathogenesis of hereditary nonpolyposis colorectal cancer (HNPCC) in humans, with mutations in hMLH1 and hMSH2 genes representing major contributors to HNPCC. Our recent work has revealed a direct physical link between hMLH1 and hMRE11 both in vitro and in vivo. Human hMRE11 represents an essential nuclease of the multifunctional protein complex (hRAD50-hMRE11-NBS1) that promotes repair of DNA double-strand breaks and plays a role in the signaling of DNA damage response as well as in meiotic recombination and telomere maintenance. Mutations in hMRE11 contribute to the development of the rare "AT-like" (ATLD) disorder. It is known that hMRE11-associated complex displays dynamic spatial and temporal cellular distribution and form DNA-damage induced foci at the sites of DNA damage. Therefore, the long-term goal of this research program is to elucidate the molecular mechanisms involved in MMR and the functional links between MMR proteins and the hMRE11-associated protein complex, as well as the biological effects of mutations that disrupt the interplay among these protein factors. We propose to address these important issues through a vigorous systematic approach that includes the following specific aims: (1) to test the hypothesis that hMRE11 represents a functional component of the DNA mismatch repair pathway; (2) to determine whether hMLH1 binding stimulates nuclease activities of hMRE11 and the functional roles of hMRE11 nuclease activities in the process of MMR; (3) to test the hypothesis that hMLH1 HNPCC missense mutations differentially affect the formation of hMLHI-hMRE11 and hMLHI-hPMS2 heterocomplexes, and therefore disruption of hMLH1-hMRE11 complex represents an alternative mechanism underlying the pathogenic effects of hMLH1 HNPCC mutations. The results of the proposed studies will provide insight into the molecular mechanisms by which MMR and the hMREll-associated complex act together to maintain genetic stability, as well as to provide a better understanding of hMLH1 and/or hMRE11 deficiencies in human diseases such as cancer.