GENOME STABILITY THROUGH BLOOM SYNDROME HELICASE AND RAD51 COMPLEX FORMATION

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。肿瘤细胞的一个标志性特征是高度不稳定的基因组。布鲁姆综合征（BS）是一种常染色体隐性遗传病，由BLM基因突变引起，表现出异常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蛋白被认为通过3-5 DNA解旋酶活性来影响基因组的稳定性，该酶活性可以稳定DNA损伤引起的停滞复制分叉。由于许多种类的基因毒性物质已被证明可以阻断复制，因此这一信息对于广泛理解细胞对基因毒性物质的反应是必不可少的。同源重组修复（HRR）途径是SCE形成和折叠复制叉恢复所必需的。因此，HRR对维持基因组稳定性至关重要。Rad51是HRR通路的核心蛋白，它与Blm发生物理相互作用，因此可能在BS细胞中SCE事件水平升高中发挥作用。本提案的目标是确定Blm的氨基酸残基与Rad51的物理相互作用，并确定该复合体作为分子开关的功能，通过该分子开关，细胞可以控制途径选择（复制叉稳定与崩溃叉的HRR恢复）。了解复合体形成的物理参数将有助于确定复合体形成的功能意义（例如，Blm解旋酶活性的增加可能导致基因组内更大的稳定性）。该实验将解决以下具体目标：1)通过从末端系统删除25个氨基酸增量来完善负责介导与Rad51复合物形成的Blm氨基酸序列；2)确定基因组不稳定性的SCE标记依赖于Blm与Rad51之间的复合物形成；3)证明Blm-Rad51复合物通过调节每种蛋白的体外活性而发挥分子开关的作用；4)评价Blm解旋酶活性与Rad51复合物形成的关系；5)评估Rad51的链位移活性作为与Blm形成复合物的函数。

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. One of the hallmark features of tumor cells is a highly unstable genome. Bloom syndrome (BS), an autosomal recessive disorder that results from a mutation of the BLM gene, exhibits extraordinarily high levels of sister chromatid exchange (SCE) events, a marker of genomic instability. Blm protein is thought to influence genome stability through the prime 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 broad 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 therefore 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 function of the complex as a molecular switch through which the cell can govern pathway choice (replication fork stabilization vs. HRR restoration of a collapsed fork). Knowledge of the physical parameters of complex formation will assist in the determination of the functional significance of complex formation (e.g. increased helicase activity of Blm could result in greater stability within the genome). 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; 4) Evaluate Blm helicase activity as a function of complex formation with Rad51; and 5) Evaluate strand displacement activity of Rad51 as a function of complex formation with Blm.