| Biochemistry
of homologous recombination 
Characterization
of recombinase function in vivo
Characterization
of DNA intermediates in meiotic recombination
Genetic dissection
of BRCA1 function
Intergenic suppression
of BRCA1 function
Characterization
of drugs that alter the efficiency of homologous recombination
Most eukaryotes use two structurally
related recombinases, Rad51 and Dmc1, to carry out meiotic recombination.
Although both of these proteins are needed for meiotic recombination,
Rad51 is sufficient to promote mitotic recombinational repair
of DNA damage. We are interested in the relationship of the functions
of Rad51 and Dmc1, during meiotic recombination. We study this
relationship using biochemical, cell biological, and molecular
genetic approaches.
General mechanism
for mediated assembly of recombinase. Single strand DNA
binding protein (SSB) binds to single stranded DNA. Mediator protein
then binds SSB making it possible for recombinase to initiate
a filament and displace SSB.
We have purified the meiosis-specific
recombinase Dmc1 as well as four accessory factors that influence
its activity in vivo. These factors include two heterodimeric
proteins Hop2-Mnd1 and Sae3-Mei5 as well as the Swi2-related DNA
translocase Tid1/Rdh54 and the multifunctional single strand DNA
binding protein RPA. We are working to reconstitute efficient
homologous recombination using these proteins and plasmid DNA
substrates. Each of the accessory factors has been found to stimulate
Dmc1’s activity when added individually to reactions. Efforts
to find conditions in which multiple factors have additive or
synergistic activity are underway.
Inhibition of Dmc1’s
ATPase activity by RPA. Dmc1’s ATPase activity
is DNA dependent. Thus, ATPase activity can be used as an indirect
measure of DNA binding in solution. This experiment shows that
RPA can outcompete Dmc1 for binding sites on ssDNA as expected
if Dmc1 requires a mediator for assembly. (Method described in
Hong et al. J Biol Chem. 2001 Nov 9;276(45):41906-12.)
In addition to looking at
conversion of DNA substrates to recombination products we monitor
assembly of Dmc1 filaments using both electron and atomic force
microscopy (AFM). Our work on AFM is being conducted in collaboration
with Dr.
Stephen Sibener of University of Chicago’s
Department of Chemistry. Our current work involves structural
comparison of filaments formed by Rad51 and Dmc1 and the effect
of accessory factors on filament structure.
Electron micrograph
of negatively stained Dmc1-ssDNA helical filaments.
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