Understanding the mechanisms that result in increased genetic instability is of great interest to those studying the development of cancer. Inhibition of DNA replication can lead to increases in the frequency of gross chromosomal rearrangements (GCRs), that can arise from DNA damage associated with stalled or collapsed replication forks. In the yeast, Schizosaccharomyces pombe, cells activate specific checkpoint pathways in response to DNA damage, and these checkpoints are essential to allow time for repair. We have discovered a novel checkpoint phenotype associated with mutants in DNA replication initiation proteins called rid (for replication initiation defective). When grown under semi-permissive conditions, rid mutants are delayed in the cell cycle but fail to activate the inter-S phase checkpoint. However, these cells still require the checkpoint kinase Chk1 for viability, suggesting an alternative checkpoint is activated. We propose to investigate the nature of the damage generated in rid mutants and to identify checkpoint/repair proteins required for rid viability. To do this, we will employ both genetic screens and gene microarray analysis. We will also determine whether mutation rates, including the incidence of GCRS, increase in the rid or checkpoint mutants. The results of the proposed experiments should provide a much better understanding of checkpoint pathways that respond to defects in the initiation of DNA replication. Considering the conserved aspects of many of these processes, our experimental findings are likely to be applicable to higher eukaryotic cells, including human. Moreover, our results will likely identify new drug targets for cancer therapy, and reveal potentially less mutagenic strategies to block cell cycle progression in cancer cells.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA099034-04
Application #
7093040
Study Section
Special Emphasis Panel (ZCA1-SRRB-3 (O1))
Program Officer
Pelroy, Richard
Project Start
2003-05-01
Project End
2008-04-30
Budget Start
2006-05-04
Budget End
2007-04-30
Support Year
4
Fiscal Year
2006
Total Cost
$309,855
Indirect Cost
Name
University of Miami School of Medicine
Department
Biochemistry
Type
Schools of Medicine
DUNS #
052780918
City
Miami
State
FL
Country
United States
Zip Code
33146
Yin, Ling; Locovei, Alexandra Monica; D'Urso, Gennaro (2008) Activation of the DNA damage checkpoint in mutants defective in DNA replication initiation. Mol Biol Cell 19:4374-82
Rodriguez-Menocal, Luis; D'Urso, Gennaro (2004) Programmed cell death in fission yeast. FEMS Yeast Res 5:111-7
Spiga, Maria-Grazia; D'Urso, Gennaro (2004) Identification and cloning of two putative subunits of DNA polymerase epsilon in fission yeast. Nucleic Acids Res 32:4945-53
Oltra, Elisa; Verde, Fulvia; Werner, Rudolf et al. (2004) A novel RING-finger-like protein Ini1 is essential for cell cycle progression in fission yeast. J Cell Sci 117:967-74
Feng, Wenyi; Rodriguez-Menocal, Luis; Tolun, Gokhan et al. (2003) Schizosacchromyces pombe Dpb2 binds to origin DNA early in S phase and is required for chromosomal DNA replication. Mol Biol Cell 14:3427-36