The molecular mechanism by which bacteriophage T7 replicates its DNA has been well characterized. All of the proteins known to have a role in the replication process have been isolated in homogeneous form and their contribution in the replication of the T7 DNA molecule have, in general, been characterized. The DNA polymerase and gene 4 protein (T7 primase) of bacteriophage T7 are able to initiate DNA synthesis at a phosphodiester bond interruption (nick) in a duplex T7 DNA molecule leading to extensive DNA synthesis. In the prosence of the T7 RNA polymerase, DNA synthesis instead can initiate at the origin of T7 DNA replication which lies between nucleotide 5,921 and 6,049. This site-specific synthesis gives rise to replication bubbles at the primary origin as seen in the electron microscope. We propose to use this well characterized system as a model to study the effects of covalent modification of a DNA template by a carcinogen. We will (i) determine and compare the consequences of benzo[a]pyrene, aminofluorene, and acetylaminofluorene adducts on RNA synthesis by the T7 RNA polymerase and DNA synthesis by the T7 DNA polymerase and gene 4 protein; (ii) determine, using DNA and RNA sequencing techniques, whether synthesis is blocked by these adducts and identify the specific sites of blockage; (iii) study the effects of DNA modification on the binding interactions between the T7 replication proteins and DNA templates; and (iv) determine the effect on the initiation of T7 DNA replication in vitro. These studies will use both single-stranded, duplex linear, and supercoiled DNA templates and DNA molecules which contain adducts specifically incorporated into only the strand serving as the template or into only the strand which is being displaced.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA035451-03
Application #
3173011
Study Section
Biochemistry Study Section (BIO)
Project Start
1983-08-01
Project End
1987-01-31
Budget Start
1986-02-01
Budget End
1987-01-31
Support Year
3
Fiscal Year
1986
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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