Effects of benzo[a]pyrene-DNA adducts on a reconstituted replication system.
We have used a partially reconstituted replication system consisting of T7 DNA polymerase and T7 gene 4 protein to examine the effect of benzo[a]pyrene (B[a]P) adducts on DNA synthesis and gene 4 protein activities. The gene 4 protein is required for T7 DNA replication because of its ability to act as both a primase and helicase. We show here that total synthesis decreases as the level of adducts per molecule of DNA increases, suggesting that the B[a]P adducts are blocking an aspect of the replication process. Polyacrylamide gels indicate that a shorter DNA product is produced on modified templates and this is confirmed by determining the average chain lengths from the ratio of chain initiations to chain elongation. Gene 4 protein primed synthesis reactions display a greater sensitivity to the presence of B[a]P adducts than do oligonucleotide-primed reactions. By challenging synthesis on oligonucleotide-primed B[a]P-modified DNA with unmodified DNA, we present evidence that the T7 DNA polymerase freely dissociates after encountering an adduct. Prior studies [Brown, W. C., & Romano, L. J. (1989) J. Biol. Chem. 264, 6748-6754] have shown that the gene 4 protein alone does not dissociate from the template during translocation upon encountering an adduct. However, when gene 4 protein primed DNA synthesis is challenged, we observe an increase in synthesis but to lesser extent than observed on oligonucleotide-primed synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
Brown
W C
WC
Department of Chemistry, Wayne State University, Detroit, Michigan 48202.
Romano
L J
LJ
D001564:Benzo(a)pyrene / Q000737:chemistry*; D002474:Cell-Free System; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry*; D004249:DNA Damage; D004265:DNA Helicases / Q000502:physiology*; D019915:DNA Primase; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000502:physiology*; D006868:Hydrolysis; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D012316:RNA Nucleotidyltransferases / Q000502:physiology*; D013604:T-Phages / Q000201:enzymology*; D013698:Templates, Genetic
D046911:Macromolecular Substances; D001564:Benzo(a)pyrene; D004247:DNA; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004259:DNA-Directed DNA Polymerase; D004265:DNA Helicases