Homologous recombination is a high-fidelity DNA repair process that promotes the integrity of the eukaryotic genome. Recombination can be initiated either by double-strand breaks (DSBs) or single-strand gaps, with individual events being resolved to produce a non-crossover or crossover product (NCO or CO, respectively). Because CO events are uniquely associated with chromosome rearrangements, regulation of the CO-NCO outcome is of particular relevance to genome stability. The proposed research will use a transformation-based, gap-repair assay and a chromosomal, HO-based assay as models for mitotic DSB repair in the yeast Saccharomyces cerevisiae. The advantages of these assays are that the position and nature of the recombination-initiating lesion are known, both CO and NCO events can be isolated, and the presence of silent sequence polymorphisms allows the molecular structure of recombination intermediates to be determined. Each of three aims will focus on understanding precisely how defined activities regulate the CO-NCO outcome.
One aim will examine the nucleases that resect the 5" strands of the broken ends, thereby generating the 3" tails required to initiate strand invasion. We propose that the extent of resection is linked to recombination pathway choice and NCO-CO outcome, and this will be tested.
A second aim will examine the Mph1, Sgs1 and Srs2 helicases, each of which independently promotes the NCO outcome during DSB repair.
A third aim will focus on the Holliday junction, the formation and resolution of which is required to generate CO products. These three aims will be accomplished by examining the extents and positions of strand transfer during individual recombination events.
A final aim will focus on understanding how the MMR machinery limits recombination between diverged sequences, an activity that is directed primarily towards CO intermediates. Basic knowledge gained from these studies will be relevant to issues of genome stability in high eukaryotes.

Public Health Relevance

Mitotic recombination is an essential process in mammalian cells, with perturbations being associated with cancer-predisposition and premature-aging syndromes. MMR defects are likewise associated with a hereditary form of cancer. Because these basic genome-stabilizing processes are highly conserved from yeast to humans, basic knowledge gained from yeast studies will be directly relevant to human health issues.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM038464-26
Application #
8499320
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Janes, Daniel E
Project Start
1987-07-01
Project End
2014-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
26
Fiscal Year
2013
Total Cost
$299,176
Indirect Cost
$103,329
Name
Duke University
Department
Genetics
Type
Schools of Medicine
DUNS #
044387793
City
Durham
State
NC
Country
United States
Zip Code
27705
Boiteux, Serge; Jinks-Robertson, Sue (2013) DNA repair mechanisms and the bypass of DNA damage in Saccharomyces cerevisiae. Genetics 193:1025-64
Mitchel, Katrina; Lehner, Kevin; Jinks-Robertson, Sue (2013) Heteroduplex DNA position defines the roles of the Sgs1, Srs2, and Mph1 helicases in promoting distinct recombination outcomes. PLoS Genet 9:e1003340
Guo, Xiaoge; Jinks-Robertson, Sue (2013) Removal of N-6-methyladenine by the nucleotide excision repair pathway triggers the repair of mismatches in yeast gap-repair intermediates. DNA Repair (Amst) 12:1053-61
Guo, Xiaoge; Jinks-Robertson, Sue (2013) Roles of exonucleases and translesion synthesis DNA polymerases during mitotic gap repair in yeast. DNA Repair (Amst) 12:1024-30
Kim, Nayun; Jinks-Robertson, Sue (2012) Transcription as a source of genome instability. Nat Rev Genet 13:204-14
Lippert, Malcolm J; Kim, Nayun; Cho, Jang-Eun et al. (2011) Role for topoisomerase 1 in transcription-associated mutagenesis in yeast. Proc Natl Acad Sci U S A 108:698-703
Kim, Nayun; Jinks-Robertson, Sue (2011) Guanine repeat-containing sequences confer transcription-dependent instability in an orientation-specific manner in yeast. DNA Repair (Amst) 10:953-60
Kim, Nayun; Huang, Shar-yin N; Williams, Jessica S et al. (2011) Mutagenic processing of ribonucleotides in DNA by yeast topoisomerase I. Science 332:1561-4
Kim, Nayun; Jinks-Robertson, Sue (2009) dUTP incorporation into genomic DNA is linked to transcription in yeast. Nature 459:1150-3
Kim, Nayun; Abdulovic, Amy L; Gealy, Regan et al. (2007) Transcription-associated mutagenesis in yeast is directly proportional to the level of gene expression and influenced by the direction of DNA replication. DNA Repair (Amst) 6:1285-96