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Genome Stability through Bloom Syndrome Helicase and Rad51 Complex
Formation (2005 - Present)
Investigator:
Karen Almeida,
Rhode Island
College
Mentor: Robert
Sobol, University of Pittsburgh
Abstract:
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 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 complex’s
function 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:
Specific Aim 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.
Specific Aim 2. Establish that the SCE marker of
genomic instability is dependent on complex formation between Blm and
Rad51.
Specific Aim 3. Demonstrate that the Blm-Rad51 complex
functions as a molecular switch by modulating the in vitro activity of
each protein.
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Evaluate Blm
helicase activity as a function of complex formation with Rad51
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Evaluate strand
displacement activity of Rad51 as a function of complex formation with
Blm
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