Research Interests

We study the roles of recombination repair proteins in genome stability in response to spontaneous DNA damage.  DNA damage has its most serious consequences during DNA replication as bulky lesions and abasic sites can stall a replication fork and a single strand nick will be converted to a double strand break (DSB) during replication and collapse the replication fork.  Both types of replication arrest require homologous recombination (HR) functions for replication restart and can trigger DNA damage checkpoint activation.  We have been focusing on the RAD54 gene, which in required for the strand invasion step of HR during D loop formation.  In addition to homologous recombination rescue of stalled replication forks, some types of spontaneous DNA damage that interfere with replication are repaired by alternative repair pathways that do not use homologous recombination.  Indeed, in these situations homologous recombination is deleterious to the cell and is actively avoided.  One mode of regulation is through the Srs2 DNA helicase regulates use of the HR pathway through reversal of an early intermediate in HR, the Rad51 filament.

1. RAD54 function in HR.
RAD54 protein functions act several steps in HR.  It has ATPase hydrolysis activity and promotes D-loop formation, the formation of heteroduplex DNA from two homologous DNA duplexes.  RAD54 also has chromatin remodeling activity in vitro and may function in removing chromatin to allow access of HR proteins to the DNA damage site.
We have used yeast as a model to assess the impact of human RAD54 mutations in gene function.  These mutations are based on those found in RAD54 genes mutated in human tumors.  Some mutants had no effect on Rad54 function in genetic and biochemical assays while other mutations completely inactivated the gene by genetic and biochemical assays.
We continue to study the role of RAD54 in HR.  Our studies implicate RAD54 in late functions, perhaps in centromere activity for mitosis.

2. Sister chromatid cohesion.
We have found that sister chromatid cohesion is necessary for HR repair of DNA damage.  We suggest that DNA damage bypass pathways that involve HR require sister chromatid cohesion in order to identify the correct partners for HR to restart collapsed replication forks.  We have found that factors associated with the replication fork are required for establishing sister chromatid cohesion.  Our goals are to show that this cohesion is established at sites of replication fork stalling.  One of the novel factors involved in sister chromatid cohesion is MRC1.  MRC1 is a DNA damage checkpoint gene and replication fork associated factor.  We are developing a model whereby MRC1 is used to help in establishing a sister chromatid cohesion complex at the site of stalled replication forks.  We are currently testing this model. 

3. Regulation of the HR pathway by the SRS2 DNA helicase

The SRS2 DNA helicase is expressed primarily in S phase.  It is modified by phosphorylation in response to DNA damage and appears to have additional modifications that may restrict its helicase activity.  It also interacts with key HR and replication proteins such as RAD51, the strand exchange protein, and PCNA, the processivity factor for DNA replication.  We are studying srs2 mutations that are altered in the modification sites or protein interaction domains to understand the role that these play in regulation of the response to DNA damage.