GENOME STABILITY THROUGH BLOOM SYNDROME HELICASE AND RAD51 COMPLEX FORMATION

通过 Bloom 综合征解旋酶和 RAD51 复合物形成实现基因组稳定性

• 批准号: 7381363
• 负责人: Karen H. Almeida
• 金额: $ 16.51万
• 依托单位: UNIVERSITY OF RHODE ISLAND
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7381363
• 关键词: GENOME; STABILITY; THROUGH; BLOOM; SYNDROME

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.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。肿瘤细胞的标志性特征之一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，这是基因组不稳定性的标志。Blm蛋白被认为通过主要的3-5 DNA解旋酶活性影响基因组稳定性，所述解旋酶活性可以稳定由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，因此该信息对于广泛了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。该提议的目标是定义与Rad 51物理相互作用的Blm的氨基酸残基，并确定复合物作为分子开关的功能，通过该分子开关细胞可以管理途径选择（复制叉稳定与塌陷叉的HRR恢复）。复合物形成的物理参数的知识将有助于确定复合物形成的功能意义（例如，Blm的解旋酶活性增加可能导致基因组内更大的稳定性）。1）通过系统性地删除末端25个氨基酸增量来优化负责介导与Rad 51形成复合物的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而作为分子开关起作用;和5）评估Rad 51的链置换活性作为与Blm形成复合物的函数。