Enzymatic DNA synthesis and its inhibition by carcinogenic DNA adducts.

摘要

In the process of DNA synthesis the DNA polymerase undergoes structural changes that are critical for its activity. In this work, complexes of Klenow fragment of E. coli DNA polymerase I with DNA primer-templates were analyzed by several biochemical approaches. The stability the enzyme-DNA complexes in both the open and closed forms was measured by a gel retardation assay. The polymerase was shown to interact with DNA more strongly in the closed conformation than in the open binary complex. Incorrect dNTPs destabilize the polymerase-DNA interaction. Interestingly, the degree of this destabilization can be directly correlated to how restricted the amount of the available space is in the active site of the polymerase.; Tryptic digestion analysis was employed to directly detect the formation of the closed complex in the presence of the next Watson-Crick nucleotide. This conformational change was also detected in the presence of the nucleotide analog that lacks the hydrogen bonding ability, but retains the geometric shape of a Watson-Crick nucleotide. This finding suggests that the hydrogen bonding between the incoming dNTP and the template is not absolutely required for the conformational change to occur. Also, this change in the structure of the DNA polymerase is inhibited by chemical modifications of the incoming nucleotides and of the DNA molecule in the active site.; Bulky DNA adducts formed by N-2-acetylaminotluorene are known to interfere with the DNA synthesis. The acetylaminofluorene (AAF) adduct is a very strong block for DNA replication in vitro, and the aminofluorene (AF) adduct is much more easily by passed. The templates containing site-specifically positioned adducts were synthesized and the complexes of Klenow fragment with the modified DNA were studied by the gel shift assay and by the nuclease and protease footprinting analyses. The results suggest that the AAF-adduct inhibits the DNA synthesis by blocking the conformational change in the structure of the DNA polymerase. The non-blocking AF adduct is much less capable of inhibiting this rearrangement. Finally, experiments with DNA adducts formed by benzo[ a]pyrene demonstrate that these bulky compounds also block the conformational change in the polymerase structure, but by a mechanism that is different from the one of the AAF adduct.