Repair of Damaged Chromosomes Mediated by the Bacterial RecN Protein

描述（由申请人提供）：本研究计划的总体目标是揭示RecN的分子功能，RecN是细菌DNA双链断裂（DSBs）修复的关键蛋白质。细菌RecN蛋白与染色体结构维持（SMC）蛋白家族具有显著的同源性。真核SMC蛋白在染色体凝聚、姐妹染色单体内聚和重组DNA修复等多种DNA代谢过程中具有重要的（尽管尚未完全了解）内护和肿瘤抑制作用。尽管大量的遗传证据强调了RecN蛋白对细菌基因组维持的重要性，但对其功能的实质性生化研究很少，因此，需要RecN的特定反应尚未确定。我们已经成功地鉴定了几种由RecN蛋白介导的体外活性，包括内聚功能和重组刺激。我们现在定位于破译重组DNA修复途径中RecN功能的机制细节，利用生物化学，生物物理和生物学方法的组合。这个项目有三个具体目标。首先，我们将进一步表征与底物结合和催化相关的RecN蛋白的生化和生物物理特性。接下来，我们将通过体外通路重构和基因筛选来确定RecN在介导重组修复中的作用。最后，我们将确定在所有细菌RecN蛋白中保守的多个序列基序的贡献，并为真核SMC同源物所共享。我们期望这项工作的结果将为理解真核SMC蛋白在基因组维持中的分子作用提供有价值的模型。

DESCRIPTION (provided by applicant): The overall goal of this research proposal is to reveal the molecular function of RecN, a protein critical to the repair of DNA double-stranded breaks (DSBs) in bacteria. Bacterial RecN proteins share significant homology to the Structural Maintenance of Chromosomes (SMC) family of proteins. Eukaryotic SMC proteins have essential (although not fully understood) housekeeping and tumor suppressor roles in a variety of DNA metabolic processes such as chromosomal condensation, sister chromatid cohesion and recombinational DNA repair. Although extensive genetic evidence underscores the importance of RecN proteins to bacterial genome maintenance, there has been little substantive biochemical investigation into their function and, consequently, the specific reactions requiring RecN have not been identified. We have successfully identified several in vitro activities mediated by the RecN protein including a cohesin function and the stimulation of recombination. We are now positioned to decipher mechanistic details of RecN function in recombinational DNA repair pathways, utilizing a combination of biochemical, biophysical and biological approaches. This project has three specific aims. First, we will further characterize the biochemical and biophysical properties of the RecN protein related to substrate binding and catalysis. Next, we will determine the role of RecN in mediating recombinational repair by using both in vitro pathway reconstitution and genetic screens. And, finally, we will determine the contribution of multiple sequence motifs conserved in all bacterial RecN proteins and shared by eukaryotic SMC orthologs. We anticipate that the results from this work will provide valuable models for understanding the molecular role that eukaryotic SMC proteins play in genome maintenance.