The nature of DNA replication origins in eukaryotic chromosomes needs to be defined before we can understand how origin activation is specified and controlled in living cells. A commitment to activate origins in cells is, at the same time, a commitment to undergo cell division and proliferation. An understanding of the mechanisms that control origin activation is essential for a full understanding of the regulation of cell proliferation and the regulatory defects that lead to uncontrolled proliferation, as in cancer. At present, chromosomally-derived origins are best defined in the yeast, S. cerevisiae, where they have been characterized as autonomously replicating sequences (ARS) in plasmids. However, little is known about the determinants of origin activity within cellular chromosomes. In this proposal, two ARS elements have been selected for further study based on their unique properties in terms of origin function within yeast chromosomes: (l) the rDNA ARS, present in each of the 100-200 ribosomal DNA repeats, only a minority of which are actually used as replication origins in the chromosome, and (2) ARS3O3, a functional ARS element in a plasmid which is silent as an origin in its native chromosomal location. We will address two major questions of general importance to the understanding of the nature of chromosomal origins and the factors that regulate their usage: (1) what is the molecular basis for the low frequency of rDNA origin usage in a chromosome? (2) what is the molecular basis for origin silencing at ARS303 in the chromosome? To address these questions, we will (a) identify the cis-acting components that are sufficient to account for the full activity of the rDNA ADS and ARS3O3 within a plasmid, (b) identify the cis-acting elements, both positive and negative, as well as other factors that govern the level of replication origin usage in a chromosome (rDNA) and the silencing of an origin within a chromosome (ARS303) and, (c) identify DNA-protein interactions and DNA structural alterations that occur in active and silent origins, and identify the cis-components required for those interactions and structural alterations. Identification of the genetic requirements for chromosomal origin function will be achieved by taking advantage of the facility with which yeast undergo homologous DNA recombination, permitting the precise replacement of a wild-type chromosomal locus with a site-specific mutant locus carried on a plasmid vector. The proposed studies are made feasible as a result of novel approaches we have devised to assay a single-copy rDNA ARS in the chromosome and to detect origin activity associated with the normally silent ARS3O3.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM030614-12A1
Application #
2175855
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1983-03-01
Project End
1999-06-30
Budget Start
1995-07-01
Budget End
1996-06-30
Support Year
12
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Roswell Park Cancer Institute Corp
Department
Type
DUNS #
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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