Activation and regulation of DNA replication are of fundamental importance to proper growth and differentiation of cells and development of organisms. Understanding how the molecular events that control DNA replication are altered by genetic mutations, environmental chemicals or therapeutic drugs is important in the prevention and treatment of diseases such as cancer. We will continue to use the yeast, Saccharomyces cerevisiae, to investigate how DNA replication is regulated in cell chromosomes. We have identified cis-acting DNA elements that are necessary and sufficient to specify and activate a chromosomal replication origin, 0RI305.
One specific aim i s to complete the characterization of a DNA unwinding element (DUE) present in 0RI305 and detected in the plasmid Autonomously Replicating Sequence, ARS3O5. We have discovered that silent replication origins near a transcriptionally-inactive chromosomal locus (HML) can be activated in the chromosome. One key component of origin silencing is the proximity of the nearest active origin in the chromosome. Our preliminary studies indicate that another important component is a chromosome position effect near HML.
A specific aim i s to identify DNA sequences and chromosome contexts responsible for the chromosome position effect that silences replication origins. Our preliminary studies indicate that, when activated, silent origins fire late in S phase.
A specific aim i s to identify the chromosome context that determines late replication timing at silent origins and to identify novel DNA elements that control late replication timing. Preliminary studies show that origin silencing is linked to the cellular response to DNA damage and that mutations in certain cell-cycle checkpoint genes activate silent origins.
A specific aim i s to examine the role of checkpoint genes and newly identified cis-acting mutations in controlling specific protein associations involved in origin silencing and late replication timing.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM030614-19
Application #
6765813
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Portnoy, Matthew
Project Start
1983-03-01
Project End
2008-06-30
Budget Start
2004-07-01
Budget End
2008-06-30
Support Year
19
Fiscal Year
2004
Total Cost
$346,617
Indirect Cost
Name
Roswell Park Cancer Institute Corp
Department
Type
DUNS #
824771034
City
Buffalo
State
NY
Country
United States
Zip Code
14263
Minca, Eugen C; Kowalski, David (2011) Replication fork stalling by bulky DNA damage: localization at active origins and checkpoint modulation. Nucleic Acids Res 39:2610-23
Minca, Eugen C; Kowalski, David (2010) Multiple Rad5 activities mediate sister chromatid recombination to bypass DNA damage at stalled replication forks. Mol Cell 38:649-61
Kowalski, David; Pendyala, Lakshmi; Daignan-Fornier, Bertrand et al. (2008) Dysregulation of purine nucleotide biosynthesis pathways modulates cisplatin cytotoxicity in Saccharomyces cerevisiae. Mol Pharmacol 74:1092-100
Huang, Ruea-Yea; Kowalski, David; Minderman, Hans et al. (2007) Small ubiquitin-related modifier pathway is a major determinant of doxorubicin cytotoxicity in Saccharomyces cerevisiae. Cancer Res 67:765-72
Huang, Ruea-Yea; Eddy, Martha; Vujcic, Marija et al. (2005) Genome-wide screen identifies genes whose inactivation confer resistance to cisplatin in Saccharomyces cerevisiae. Cancer Res 65:5890-7
Dziegielewska, Barbara; Kowalski, David; Beerman, Terry A (2004) SV40 DNA replication inhibition by the monofunctional DNA alkylator Et743. Biochemistry 43:14228-37
Huang, Yanlin; Kowalski, David (2004) PATTERNFINDER: combined analysis of DNA regulatory sequences and double-helix stability. BMC Bioinformatics 5:134
Huang, Yanlin; Kowalski, David (2003) WEB-THERMODYN: Sequence analysis software for profiling DNA helical stability. Nucleic Acids Res 31:3819-21
Wang, Y; Vujcic, M; Kowalski, D (2001) DNA replication forks pause at silent origins near the HML locus in budding yeast. Mol Cell Biol 21:4938-48
Wang, Y; Beerman, T A; Kowalski, D (2001) Antitumor drug adozelesin differentially affects active and silent origins of DNA replication in yeast checkpoint kinase mutants. Cancer Res 61:3787-94

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