DNA polymerase, RNA polymerase and exonuclease activities on a DNA sequence modified by benzo[a]pyrene diolepoxide.
Adducts produced by modification of DNA with benzo[a]pyrene diolepoxide (BPDE) are known to inhibit both DNA and RNA synthesis. This phenomenon has been used as a method for determining the distribution of carcinogen binding within defined DNA sequences. A critical comparison of different enzyme activities on adducted DNA is needed, since different enzymes may process adducted DNA differently. Thus, we compared blocks in DNA polymerase activity with that of an RNA polymerase and with an exonuclease at single base resolution. BPDE adducts blocked the progression of cloned T7 DNA polymerase (Sequenase) in a dose-dependent manner. Although the majority of these blocks were at one base prior to adducted guanines, we also observed some blocks opposite specific guanines, suggesting that in some sequences the polymerase inserted a base opposite the modified guanine. Digestion with T4 DNA polymerase (3'----5') exonuclease activity was also blocked in BPDE-adducted DNA; however, fragments produced by blocks in T4 exonuclease migrated two or more bases longer than the corresponding guanine. Mapping of adduct distributions using both Sequenase and T4 exonuclease gave similar results, demonstrating that a long tract of guanines was preferentially modified, and within a polyguanine sequence, the 5' guanines were more heavily modified than the 3' guanines. Transcription of adducted DNA by SP6 RNA polymerase was also inhibited in a dose-dependent manner. However, adducted bases which posed strong blocks to the DNA polymerase were not always strong blocks to the RNA polymerase. Thus, in terms of adduct distribution, Sequenase and T4 exonuclease provided more consistent results than the RNA polymerase, since blockage of the RNA polymerase correlated poorly with guanines.
Thrall
B D
BD
Pacific Northwest Laboratory, Biology and Chemistry Department, Richland, WA 99352.
Mann
D B
DB
Smerdon
M J
MJ
Springer
D L
DL
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000633:toxicity*; D000818:Animals; D004247:DNA / Q000187:drug effects / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005092:Exonucleases / Q000378:metabolism*; D010957:Plasmids; D014158:Transcription, Genetic; D014981:Xenopus
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases