Regulation of Homologous Recombination in Human Cells

描述（由申请人提供）：同源重组（Homologous recombination， HR）是维持细胞基因组完整性的重要机制，通过修复双链断裂和修复受损的复制叉。RAD51重组酶催化HR通路的中心步骤，通过同源依赖的链入侵形成联合分子。BLM解旋酶是Bloom's综合征（BS）中发生突变的基因，调控RAD51。BLM可以促进RAD51的功能，例如通过生成与RAD51结合的底物，或者通过解绕RAD51催化产物来抑制RAD51的功能，从而阻止有毒重组中间体的积累。因此，通过调控RAD51， BLM在HR中具有促重组和抗重组的功能。我们已经证明BLM被小的泛素相关修饰物（sumo）修饰，并且BLM sumo化调节BLM在停滞复制分叉处的功能，刺激HR修复。此外，RAD51与sumo结合，BLM summoylation刺激BLM与RAD51的相互作用。我们的研究结果支持了一个模型，在这个模型中，BLM的summoylation控制着BLM的抗重组和促重组功能之间的切换。为了验证这一假设，我们有四个具体目标：(1)表征BLM SUMOylation在稳定复制分叉中的作用；(2) RAD51 SUMO结合在复制叉稳定性和HR修复中的作用表征；(3)分析BLM SUMOylation对BLM生化活性和RAD51功能的影响；(4)分析BLM SUMO化和RAD51 SUMO结合对基因组完整性的影响。我们的研究将阐明在受损复制分叉处调节HR的分子机制，为BLM和RAD51在HR修复中的动态功能提供见解，并进一步了解SUMO途径如何调节这些功能。由于癌细胞中的HR修复机制经常失调，我们的工作将导致对癌症基因组不稳定性的更好理解，并将促进这种不稳定性在癌症治疗中的利用。

DESCRIPTION (provided by applicant): Homologous recombination (HR) is an essential mechanism that maintains genomic integrity in cells by repairing double-strand breaks and repairing damaged replication forks. The RAD51 recombinase catalyzes the central step in the HR pathway, forming joint molecules through homology-dependent strand invasion. The BLM helicase, which is the gene mutated in Bloom's syndrome (BS), regulates RAD51. BLM can promote RAD51 function, for example by generating substrate for RAD51 binding, or it can inhibit RAD51 function by unwinding the products of RAD51 catalysis, which prevents the accumulation of toxic recombination intermediates. Thus, through its regulation of RAD51, BLM has both pro- and anti-recombinogenic functions in HR. We have shown that BLM is modified by small ubiquitin-related modifiers (SUMOs) and that BLM SUMOylation regulates BLM's functions at stalled replication forks, stimulating HR repair. In addition, RAD51 is SUMO-binding, and BLM SUMOylation stimulates BLM's interaction with RAD51 in vitro. Our findings support a model in which BLM SUMOylation controls a switch between BLM's anti- recombinogenic and pro-recombinogenic functions. To test this hypothesis, we have four specific aims: (1) characterization of the role of BLM SUMOylation in stabilization of replication forks; (2) characterization of the role of RAD51 SUMO binding in replication fork stability and HR repair; (3) analysis of the effects of BLM SUMOylation on BLM's biochemical activities and RAD51 function; and (4) analysis of the effects of BLM SUMOylation and RAD51 SUMO binding in genomic integrity. Our studies will elucidate the molecular mechanisms that regulate HR at damaged replication forks, providing insights into the dynamic functions of BLM and RAD51 in HR repair and leading to a deeper understanding of how these functions are regulated by the SUMO pathway. Because HR repair mechanisms are often dysregulated in cancer cells, our work will lead to a better understanding of genomic instability in cancer and will facilitate the exploitation of this instability in cancer treatments.