Regulation of Homologous Recombination in Human Cells

描述(申请人提供)：同源重组(HR)是一种基本的机制，通过修复双链断裂和修复受损的复制叉来维持细胞基因组的完整性。RAD51重组酶催化HR途径的中心步骤，通过同源依赖的链入侵形成连接分子。BLM解旋酶是在Bloom综合征(BS)中突变的基因，它调节RAD51。BLM可以促进RAD51的功能，例如通过产生RAD51结合的底物，或者它可以通过解离RAD51催化的产物来抑制RAD51的功能，从而防止有毒重组中间体的积累。因此，BLM通过对RAD51的调节，在HR中既具有促重组功能，又具有抗重组功能。我们已经证明BLM被小泛素相关修饰物(SUMO)修饰，BLM SUMO化调节BLM在停滞的复制叉处的功能，刺激HR修复。此外，RAD51是相扑结合的，在体外，BLM的SUMO化刺激了BLM与RAD51的相互作用。我们的发现支持一种模型，在该模型中，BLM SUMO化控制着BLM的反重组和促重组功能之间的切换。为了验证这一假说，我们有四个具体的目标：(1)BLM SUMO化在复制叉子稳定中的作用；(2)RAD51相扑结合在复制叉稳定性和HR修复中的作用；(3)分析BLM SUMO化对BLM生化活性和RAD51功能的影响；以及(4)分析BLM SUMO化和RAD51相扑结合对基因组完整性的影响。我们的研究将阐明在受损的复制叉处调节HR的分子机制，为BLM和RAD51在HR修复中的动态功能提供见解，并导致对这些功能如何受到相扑通路的更深层次的理解。由于HR修复机制经常在癌细胞中失控，我们的工作将有助于更好地理解癌症中的基因组不稳定性，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.