GENOME STABILITY THROUGH BLOOM SYNDROME HELICASE AND RAD51 COMPLEX FORMATION

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 肿瘤细胞的标志性特征是高度不稳定的基因组。Bloom综合征（BS）是一种常染色体隐性遗传疾病，由BLM基因突变引起，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个途径影响基因组稳定性-5 DNA解旋酶的活性，可以稳定由DNA损伤引起的停滞的复制叉。由于许多类别的遗传毒性剂已被证明可以阻断复制，因此该信息对于理解细胞对遗传毒性剂的反应至关重要。同源重组修复（HRR）途径是SCE形成和恢复折叠的复制叉所必需的。因此，HRR在维持基因组稳定性方面至关重要。Rad 51是HRR途径的核心蛋白，与BLM发生物理相互作用，因此可能在BS细胞中观察到的SCE事件水平升高中发挥作用。该建议的目的是确定与Rad 51物理相互作用的BLM的氨基酸残基，并确定复合物它的功能就像一个分子开关，通过它细胞可以控制通路的选择。物理参数的知识将有助于确定复杂地层的功能意义。所提出的实验将解决以下具体目标：（1）。通过从末端系统性缺失25个氨基酸增量来细化负责介导与Rad 51形成复合物的BLM氨基酸序列;（2.）建立基因组不稳定性的SCE标记依赖于BLM和Rad 51之间的复合物形成;（3）证明BLM-Rad 51复合物通过调节每种蛋白质的体外活性作为分子开关发挥作用。 我们将评估BLM解旋酶活性作为Rad 51伙伴关系的函数和Rad 51链置换活性作为BLM伙伴关系的函数。

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 complexs 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 Specific 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; (2.) Establish that the SCE marker of genomic instability is dependent on complex formation between BLM and Rad51; (3.) Demonstrate that the BLM-Rad51 complex functions as a molecular switch by modulating the in vitro activity of each protein. We will evaluate BLM helicase activity as a function of Rad51 partnership and strand displacement activity of Rad51 as a function of BLM partnership.