A central tenet of eukaryotic cell biology is that DNA replication must be tightly controlled so that it occurs only once per cell cycle. We study replication control in budding yeast because this model system offers an exceptional opportunity to dissect the complex, overlapping mechanisms that are required to achieve this control with such extraordinary fidelity. Additionally, the molecular genetic tools available in budding yeast allow us to apply both simple and sophisticated technologies to query the effects of disrupting replication control. Our longstanding work has been instrumental in revealing multiple overlapping mechanisms used by eukaryotes to reduce the probability of re-initiation within a cell cycle. However, since eukaryotic genomes contains thousands of replication origins, it may be impossible to eliminate rare, sporadic re-initiation over the course of multiple cell divisions, and this problem can be significantly exacerbated by just a slight compromise in replication control. Thus one of our long-term goals is to discern the role of re-replication as a driver of genome plasticity and instability. Recently we have shown that loss of replication control is a powerful instigator of genetic alteration and variation in eukaryotes. We find that inappropriate re-initiation of DNA replication within a cell cycle can induce tandem intrachromosomal amplifications and aneuploidy by up to 104-106 folds. Here we propose to (1) investigate the mechanism of re-replication induced gene amplification; (2) investigate the mechanism of re- replication induced aneuploidy; (3) expand our ability to detect re-replication and genetic alterations arising from re-replication; and (4) explore whether re-replication provides a source of spontaneous segmental amplification during normal cellular growth. This work will establish re-replication as a potential new source of genetic variation and alteration in budding yeast that could ultimately impact how we think about molecular evolution and oncogenesis.

Public Health Relevance

Our studies in budding yeast suggest that the unscheduled re-replication of DNA could be a source of genetic alterations. Our proposed project will investigate how re-replication can cause two specific types of alterations frequently seen in cancer, gene amplification and chromosome aneuploidy, and explores whether re-replication could also contribute to the gene duplication critical for molecular evolution.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM059704-15
Application #
9263947
Study Section
Cellular Signaling and Regulatory Systems Study Section (CSRS)
Program Officer
Reddy, Michael K
Project Start
2000-07-01
Project End
2020-04-30
Budget Start
2017-05-01
Budget End
2018-04-30
Support Year
15
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of California San Francisco
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94118
Hanlon, Stacey L; Li, Joachim J (2015) Re-replication of a centromere induces chromosomal instability and aneuploidy. PLoS Genet 11:e1005039
Richardson, Christopher D; Li, Joachim J (2014) Regulatory mechanisms that prevent re-initiation of DNA replication can be locally modulated at origins by nearby sequence elements. PLoS Genet 10:e1004358
Finn, Kenneth J; Li, Joachim J (2013) Single-stranded annealing induced by re-initiation of replication origins provides a novel and efficient mechanism for generating copy number expansion via non-allelic homologous recombination. PLoS Genet 9:e1003192
Green, Brian M; Finn, Kenneth J; Li, Joachim J (2010) Loss of DNA replication control is a potent inducer of gene amplification. Science 329:943-6
Moses, Alan M; Liku, Muluye E; Li, Joachim J et al. (2007) Regulatory evolution in proteins by turnover and lineage-specific changes of cyclin-dependent kinase consensus sites. Proc Natl Acad Sci U S A 104:17713-8
Green, Brian M; Morreale, Richard J; Ozaydin, Bilge et al. (2006) Genome-wide mapping of DNA synthesis in Saccharomyces cerevisiae reveals that mechanisms preventing reinitiation of DNA replication are not redundant. Mol Biol Cell 17:2401-14
Liku, Muluye E; Nguyen, Van Q; Rosales, Audrey W et al. (2005) CDK phosphorylation of a novel NLS-NES module distributed between two subunits of the Mcm2-7 complex prevents chromosomal rereplication. Mol Biol Cell 16:5026-39
Green, Brian M; Li, Joachim J (2005) Loss of rereplication control in Saccharomyces cerevisiae results in extensive DNA damage. Mol Biol Cell 16:421-32