The long term goal of this proposal is to understand how the structure and energetics of the DNA template contribute to regulatory mechanisms for the initiation of DNA replication and transcription in living cells. Supercoiling of the DNA template is energetically unfavorable and can result in localized opening or unwinding of the helix in vitro. This proposal is a continuation of our characterization of the single-strand- specific endonucleases, mung bean nuclease and P1 nuclease, as probes for unwinding in naturally-occurring DNA sequences present in supercoiled plasmids and viral genomes. The enzymes recognize Z-DNA, cruciforms and a novel, stably-unwound DNA conformation call the AT-rich structure. The AT- rich structure occurs in several prokaryotic and eukaryotic replication origins. Extensive mutational analysis of a yeast replication origin revealed that detection of the AT-rich structure in vitro correlates with the ability to initiate DNA replication in vivo.
The specific aims of this proposal are to 1) examine the architecture, DNA sequence selectivity and energetics of the AT-rich structure, 2) investigate easily-unwound DNA sequences as determinants for initiation of replication in vivo, and 3) probe for localized DNA unwinding in chromatin. The hypothesis that the AT-rich structure is not melted but rather a lower energy DNA conformation which is partially untwisted and base paired, will be tested by 1) two dimensional gel electrophoresis of topoisomers to assess the energy and extent of DNA unwinding, and 2) analysis of DNA sequences which react with the enzymes as well as chemical probes for paired or unpaired bases. They hypothesis that the low energy of DNA unwinding is a determinant of replication initiation in vivo will be tested by comparing the effects of mutations on formation of an AT-rich structure in the chromosomal origin (oriC) of E. coil and the simian virus 40 (SV40) origin in vitro with the ability of the mutant derivatives to initiate replication in vivo. Finally, we will establish the use of mung bean nuclease and P1 nuclease as chromatin probes for unpaired and non-Watson-Crick paired bases, in SV40 chromatin at nucleotide level resolution.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM030614-08
Application #
3278415
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1983-03-01
Project End
1994-02-28
Budget Start
1990-03-01
Budget End
1991-02-28
Support Year
8
Fiscal Year
1990
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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