Homologous DNA recombination plays an essential role in repairing double strand breaks and stabilizing stalled replication forks. Defects in this process may have fatal consequences or result in chromosomal rearrangements typical of those found in tumor cells. Here we will explore the role of MMS4-MUS81 in recombination-mediated DNA repair by applying a combination of biochemical, molecular, and genetic approaches in yeast. Mms4-Mus81 was identified in yeast as a structure-specific endonuclease that functionally overlapped with the helicase-topoisomerase complex, Sgs1Top3. Our working hypothesis is that Mms4-Mus81 mediates a form of repair specific to arrested replication forks.
The first Aim applies a number of biochemical approaches to determine the nuclease's structure, regulation and substrate-specificity. The native complex will be purified from yeast extracts, assayed for nuclease activities, and co-purifying proteins will be identified and characterized. Induction of the protein by DNA damage and the role of phosphorylation will also be examined. The range of substrates recognized by the nuclease will be determined by designing and assaying substrates of varying size. In the second Aim we will determine the role of MMS4-MUS81 in a variety of recombination assays. Functional overlap with the homologous endonuclease Radl-10 will be investigated using epistasis analysis and suppression by overexpression. Chromatin immunoprecipitation will be used to to search for the presence of Mms4-Mus81 at sites of DNA repair and replication fork pausing in-vivo.
The third Aim employs several genetic screens to identify new recombinational repair genes. We will identify the pathway responsible for bypassing the SGS1 MUS81 requirement by performing an SGS1 MUS81 RAD51 synthetic-lethal screen. We will also search for suppressors of mus81' s DNA damage sensitivity. Finally, we will use a novel variation of the synthetic-lethal screen to identify mutants that require MMS4-MUS81 for double strand break repair. It is expected that the results of these experiments will shed light on the nature of replication fork arrest and the mechanism of recombination-mediated DNA repair in eukaryotes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM067956-02
Application #
6744051
Study Section
Chemical Pathology Study Section (CPA)
Program Officer
Wolfe, Paul B
Project Start
2003-05-01
Project End
2007-04-30
Budget Start
2004-05-01
Budget End
2005-04-30
Support Year
2
Fiscal Year
2004
Total Cost
$277,403
Indirect Cost
Name
Rutgers University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
001912864
City
New Brunswick
State
NJ
Country
United States
Zip Code
08901
Trowbridge, Kirsten; McKim, Kim; Brill, Steven J et al. (2007) Synthetic lethality of Drosophila in the absence of the MUS81 endonuclease and the DmBlm helicase is associated with elevated apoptosis. Genetics 176:1993-2001
Ii, Miki; Ii, Tatsuya; Brill, Steven J (2007) Mus81 functions in the quality control of replication forks at the rDNA and is involved in the maintenance of rDNA repeat number in Saccharomyces cerevisiae. Mutat Res 625:1-19
Roberts, Tania M; Kobor, Michael S; Bastin-Shanower, Suzanne A et al. (2006) Slx4 regulates DNA damage checkpoint-dependent phosphorylation of the BRCT domain protein Rtt107/Esc4. Mol Biol Cell 17:539-48
Fricke, William M; Bastin-Shanower, Suzanne A; Brill, Steven J (2005) Substrate specificity of the Saccharomyces cerevisiae Mus81-Mms4 endonuclease. DNA Repair (Amst) 4:243-51
Ii, Miki; Brill, Steven J (2005) Roles of SGS1, MUS81, and RAD51 in the repair of lagging-strand replication defects in Saccharomyces cerevisiae. Curr Genet 48:213-25
Hollingsworth, Nancy M; Brill, Steven J (2004) The Mus81 solution to resolution: generating meiotic crossovers without Holliday junctions. Genes Dev 18:117-25