The efficiency of translesion synthesis past single styrene oxide DNA adducts in vitro is polymerase-specific.
In order to examine the effect of adenine N6 adducts of styrene oxide (SO) on DNA replication, 33-mer templates were constructed bearing site-specific and stereospecific SO modifications. Both R- and S-SO adducts were introduced at four different base positions within a sequence containing codons 60-62 from the human N-ras gene. The resulting eight templates were replicated in primer extension assays using the Klenow fragment, Sequenase 2.0, T4 polymerase holoenzyme, polymerase alpha, and polymerase beta. Replication of the damaged templates was analyzed under conditions defining single and/or multiple encounters between the polymerase and the substrate. Polymerization by all five enzymes was sensitive to both the local sequence context and the chirality of the SO adduct. For example, R-SO lesions placed at the third position of N-ras codon 61 were readily bypassed, whereas stereochemically-identical lesions in other sequence contexts were often poor substrates for replication. Similarly, R- and S-SO adducts introduced within identical sequences were often bypassed nonequivalently. Significantly, the degree of adduct-directed termination and translesion synthesis during replication was also dependent on the choice of polymerase. Although SO adducts directed termination either opposite the lesion or 1 base 3' to the damage using all five polymerases, templates that were poor substrates for bypass synthesis with one enzyme were often read-through much more efficiently when a different polymerase was used. Thus, the activities of these enzymes on the SO-modified substrates produced replication profiles, or "fingerprints", that were unique to each polymerase.
Latham
G J
GJ
Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Harris
C M
CM
Harris
T M
TM
Lloyd
R S
RS
D000818:Animals; D017122:Bacteriophage T4 / Q000201:enzymology; D001483:Base Sequence; D018736:DNA Adducts / Q000633:toxicity*; D004256:DNA Polymerase I / Q000096:biosynthesis; D004257:DNA Polymerase II / Q000096:biosynthesis; D004261:DNA Replication / Q000187:drug effects*; D004259:DNA-Directed DNA Polymerase / Q000096:biosynthesis* / Q000187:drug effects; D004852:Epoxy Compounds / Q000633:toxicity*; D011905:Genes, ras; D006801:Humans; D008969:Molecular Sequence Data; D051381:Rats; D013698:Templates, Genetic; D014764:Viral Proteins / Q000096:biosynthesis
D018736:DNA Adducts; D004852:Epoxy Compounds; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; C013690:styrene oxide; D004256:DNA Polymerase I; D004257:DNA Polymerase II; D004259:DNA-Directed DNA Polymerase