GENOME STABILITY THROUGH BLOOM SYNDROME HELICASE AND RAD51 COMPLEX FORMATION

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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.