单分子技术解析BLM解旋酶在DNA同源重组修复中的分子机制及功能

DNA受到内源代谢产物以及外源物质攻击会造成多种不同的损伤，其中DNA双链断裂是最严重的损伤之一。双链断裂的DNA若不能及时正确的修复，会造成有害的染色体重组与基因突变，进而诱发疾病的产生。同源重组修复是DNA双链断裂损伤中一种能够保持基因组完整性的高精度修复方式。在此过程中，BLM解旋酶通过解旋双链DNA发挥促重组的作用，但又能与其它蛋白协作发挥抗重组功能，帮助避免不良同源重组事件的发生，然而关于BLM抗重组功能的确切机制依然是未知的。因此本项目计划通过单分子荧光光镊技术对具有多重生物学功能的BLM进行深入研究，重点讨论BLM与单链结合蛋白RPA之间的协同作用如何调控同源重组修复过程中核蛋白丝的组装与解离，以及是否破坏同源重组修复过程中已经形成的D-loop结构。通过对BLM抗重组功能的单分子水平研究，阐明BLM发挥抗重组功能的动态分子机制。

DNA is attacked by a variety of endogenous metabolites and exogenous substances to cause a variety of different damages. Among them, DSB (double-stranded break) is one of the most serious damage. If DSB cannot be repaired in time and correctly, it will cause harmful chromosomal recombination and gene mutation, and then induce disease. HR (Homologous recombination) repair is a high-precision repair method for DSB that can maintain genome integrity. During the homologous recombination repair process, the BLM helicase (Bloom syndrome helicase) is involved in the production of long 3' single-stranded DNA overhangs, which are first coated with RPA (Replication protein A). Subsequently, RPA was replaced by the recombinase RAD51 to form RAD51-ssDNA nucleoprotein filament (NPF). which were matched with homologous DNA to form a displacement D-loop for subsequent recombination and repair processes. BLM helicase plays a role in promoting recombination by unwinding double-stranded DNA, but can also cooperate with other proteins to play an anti-recombination function, helping to avoid adverse homologous recombination events. However, the exact mechanism of BLM anti-recombination function is still unknown. Therefore, this project plans to conduct in-depth research on BLM through optical tweezers with single-molecule fluorescence. Our study mainly focus on how the BLM and RPA can regulate the dissociation of RAD51 from NPF, and whether break the formed D-loop structure during HR repair. Through the single molecule research on the BLM, it provides the dynamic molecular mechanism of the BLM's anti-recombination role.