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DNA polymerase, RNA polymerase and exonuclease activities on a DNA sequence modified by benzo[a]pyrene diolepoxide
B D Thrall1, D B Mann, M J Smerdon
1Pacific Northwest Laboratory, Biology and Chemistry Department, Richland, WA 99352.
Abstract:
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.
Insights
Benzo[a]pyrene diolepoxide (BPDE) DNA adducts inhibit DNA and RNA synthesis. Comparing enzymes revealed DNA polymerases and exonucleases offer more reliable carcinogen mapping than RNA polymerase.
Area of Science:
- Biochemistry
- Molecular Biology
- Toxicology
Background:
- Benzo[a]pyrene diolepoxide (BPDE) forms DNA adducts that impede DNA and RNA synthesis.
- Understanding carcinogen binding distribution is crucial for assessing DNA damage.
- Different enzymes may process adducted DNA uniquely, necessitating comparative studies.
Purpose of the Study:
- To compare the effects of DNA polymerase, RNA polymerase, and exonuclease activities on BPDE-adducted DNA at single-base resolution.
- To evaluate the utility of different enzymes for mapping the distribution of BPDE adducts in DNA sequences.
Main Methods:
- Utilized cloned T7 DNA polymerase (Sequenase) to assess polymerase activity on BPDE-adducted DNA.
- Employed T4 DNA polymerase (3'→5') exonuclease activity for digestion of adducted DNA.
- Investigated transcription inhibition using SP6 RNA polymerase on BPDE-adducted DNA.
Main Results:
- BPDE adducts caused dose-dependent blocks in T7 DNA polymerase progression, primarily upstream of guanine adducts.
- T4 DNA polymerase exonuclease activity was also blocked, producing fragments indicating adduct positions.
- Mapping with Sequenase and T4 exonuclease revealed preferential modification of guanine tracts, with 5' guanines being more affected.
- SP6 RNA polymerase transcription was inhibited, but blocks did not consistently correlate with guanine adducts.
Conclusions:
- Sequenase and T4 exonuclease provide more consistent and reliable mapping of BPDE adduct distribution compared to SP6 RNA polymerase.
- Enzyme choice is critical for accurate determination of carcinogen binding sites in DNA.
- BPDE preferentially modifies guanine-rich regions, with specific sequence contexts influencing modification patterns.