The objective is to continue to investigate the detailed effects of the bulky carcinogenic adducts, 2-aminofluorene (AF), N-acetyl-2-aminofluorene (AAF), and benzo(a)pyrene (BP), on DNA replication. Our approach has been to study the effect of these adducts on not just a DNA polymerase alone, but, instead, to determine how a purified in vitro replication system responds to this type of damage. We propose to determine how the ancillary proteins, required for DNA replication in vivo, affect synthesis using single- or double-stranded DNA templates containing carcinogenic DNA adducts and to characterize the effect of such adducts on the fidelity of DNA synthesis, DNA unwinding, RNA transcription, DNA initiation, and protein-nucleic acid interactions. The mechanism by which E. coli single stranded DNA binding protein (SSB) stimulates DNA synthesis by the T7 DNA polymerase on native and carcinogen-modified DNA will be determined and this effect will be compared with the results obtained with other DNA polymerases. These experiments will utilize not only randomly modified single- and double-stranded DNA templates, but also DNA containing a site-specific AF and AAF adducts, which we have recently prepared. Using these site-specifically modified templates, the frequency of misincorporation during trans-lesion synthesis by DNA polymerases in the presence and absence of SSB or other single-stranded DNA binding proteins will be determined. DNA synthesis by the T7 DNA polymerase and gene 4 protein on double-stranded DNA containing AF, AAF, and BP adducts will be characterized and these studies will be subsequently extended to site-specifically modified templates. DNA molecules containing a T7 class III RNA polymerase promoter and containing an AF or AAF adduct located a unique site will be prepared and used to further characterize the effect of DNA adducts on RNA synthesis and on the initiation of DNA replication by the T7 RNA polymerase, T7 DNA polymerase, and gene 4 protein. Finally, we will determine the effect of DNA adducts on the interactions between DNA and single-stranded DNA binding proteins, T7 RNA polymerase, or the Uvr ABC endonuclease.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA035451-06
Application #
3173013
Study Section
Chemical Pathology Study Section (CPA)
Project Start
1983-08-01
Project End
1991-01-31
Budget Start
1989-02-01
Budget End
1991-01-31
Support Year
6
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Wayne State University
Department
Type
Schools of Arts and Sciences
DUNS #
City
Detroit
State
MI
Country
United States
Zip Code
48202
Yong, Y; Romano, L J (1996) Benzo[a]pyrene-DNA adducts inhibit the DNA helicase activity of the bacteriophage T7 gene 4 protein. Chem Res Toxicol 9:179-87
Yong, Y; Romano, L J (1995) Nucleotide and DNA-induced conformational changes in the bacteriophage T7 gene 4 protein. J Biol Chem 270:24509-17
Rigler, M N; Romano, L J (1995) Differences in the mechanism of stimulation of T7 DNA polymerase by two binding modes of Escherichia coli single-stranded DNA-binding protein. J Biol Chem 270:8910-9
Michaels, M L; Reid, T M; King, C M et al. (1991) Accurate in vitro translesion synthesis by Escherichia coli DNA polymerase I (large fragment) on a site-specific, aminofluorene-modified oligonucleotide. Carcinogenesis 12:1641-6
Nath, S T; Romano, L J; Romana, L J (1991) Transcription by T7 RNA polymerase using benzo[a]pyrene-modified templates. Carcinogenesis 12:973-6
Brown, W C; Romano, L J (1991) Effects of benzo[a]pyrene-DNA adducts on a reconstituted replication system. Biochemistry 30:1342-50
Brown, W C; Romano, L J (1989) Benzo[a]pyrene-DNA adducts inhibit translocation by the gene 4 protein of bacteriophage T7. J Biol Chem 264:6748-54
Myers, T W; Romano, L J (1988) Mechanism of stimulation of T7 DNA polymerase by Escherichia coli single-stranded DNA binding protein (SSB). J Biol Chem 263:17006-15
Michaels, M L; Johnson, D L; Reid, T M et al. (1987) Evidence for in vitro translesion DNA synthesis past a site-specific aminofluorene adduct. J Biol Chem 262:14648-54
Nath, S T; Lee, M S; Romano, L J (1987) Effect of carcinogenic adducts on transcription by T7 RNA polymerase. Nucleic Acids Res 15:4257-71

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