Repair of Damaged Chromosomes Mediated by the Bacterial RecN Protein

描述（由申请人提供）：本研究提案的总体目标是揭示RecN的分子功能，RecN是一种对细菌中DNA双链断裂（DSB）修复至关重要的蛋白质。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蛋白在多种DNA代谢过程中具有基本的（尽管尚未完全理解）管家和肿瘤抑制作用，如染色体凝聚、姐妹染色单体凝聚和重组DNA修复。虽然广泛的遗传证据强调了RecN蛋白对细菌基因组维护的重要性，但对其功能的实质性生化研究很少，因此，尚未确定需要RecN的特定反应。我们已经成功地确定了几个在体外活动介导的RecN蛋白，包括一个粘连蛋白的功能和刺激重组。我们现在定位于破译RecN在重组DNA修复途径中功能的机制细节，利用生物化学，生物物理和生物学方法的组合。该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蛋白的生物化学和生物物理特性。接下来，我们将通过使用体外途径重建和遗传筛选来确定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蛋白中保守并由真核SMC直系同源物共享的多个序列基序的贡献。我们预计，这项工作的结果将提供有价值的模型，了解真核SMC蛋白在基因组维护中发挥的分子作用。

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.