GENOME STABILITY THROUGH BLOOM SYNDROME HELICASE AND RAD51 COMPLEX FORMATION

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 肿瘤细胞的显著特征是高度不稳定的基因组。Bloom综合征(BS)是一种由BLM基因突变引起的常染色体隐性遗传病，表现出极高水平的姐妹染色单体交换(SCE)事件，这是基因组不稳定的标志。BLM蛋白可能通过3‘-5’DNA解旋酶的活性来影响基因组的稳定性，这种解旋酶可以稳定DNA损伤导致的复制叉停滞。由于许多类别的基因毒剂已被证明可以阻止复制，这一信息对于了解细胞对基因毒剂的反应是必不可少的。同源重组修复(HRR)途径是姐妹染色单体交换形成和修复折叠复制叉所必需的。因此，HRR在维持基因组稳定性方面是必不可少的。RAD51是HRR途径的核心蛋白，与BLM在物理上相互作用，因此可能在BS细胞中出现的姐妹染色单体交换事件水平升高中发挥作用。这项建议的目标是定义BLM与RAD51物理相互作用的氨基酸残基，并确定该复合体作为分子开关的功能，通过该分子开关，细胞可以控制途径的选择。物理参数的知识将有助于确定复合体形成的功能意义。所提出的实验将达到以下目的：(1)通过系统删除末端25个氨基酸的增量来精炼负责与RAD51形成复合物的BLM氨基酸序列；(2)测量BLM-RAD51复合物形成的生化动力学。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The hallmark feature of tumor cells is a highly unstable genome. Bloom syndrome (BS), an autosomal recessive disorder that results from mutation of the BLM gene, exhibits extraordinarily high levels of sister chromatid exchange (SCE) events, a marker of genomic instability. BLM protein may influence genome stability through 3'-5' DNA helicase activity that can stabilize stalled replication forks caused by damage to the DNA. Since many classes of genotoxic agents have been shown to block replication, this information is essential to the understanding of the cellular responses to genotoxic agents. The homologous recombinational repair (HRR) pathway is required for SCE formation and restoration of a collapsed replication fork. Therefore, HRR is essential in maintaining genomic stability. Rad51, a protein central to the HRR pathway, physically interacts with BLM and thus could play a role in the elevated levels of SCE events seen in BS cells. The goal of this proposal is to define the amino acid residues of BLM physically interacting with Rad51 and to determine the complex's function as a molecular switch through which the cell can govern pathway choice. Knowledge of the physical parameters will assist in the determination of the functional significance of complex formation. The experiments proposed will address the following aims: (1) Refine the BLM amino acid sequence responsible for mediating complex formation with Rad51 through systematic deletion of 25 amino acid increments from the termini; and (2) Measure the biochemical kinetics of BLM-Rad51 complex formation.