Effects of benzo[a]pyrene DNA adducts on Escherichia coli DNA polymerase I (Klenow fragment) primer-template interactions: evidence for inhibition of the catalytically active ternary complex formation.

Alekseyev YO, Dzantiev L, Romano LJ
Biochemistry (2001), Volume 40, Page 2282
PubMed entry


Benzo[a]pyrene diol epoxide (B[a]PDE) adducts are strong blocks of DNA ...
Benzo[a]pyrene diol epoxide (B[a]PDE) adducts are strong blocks of DNA replication in vitro, allowing the rare incorporation of a nucleotide across from the lesion and negligibly small extent of further bypass. To study the mechanistic details of this process, a gel-retardation assay was used to measure the dissociation constants for the binding of DNA polymerase I (Klenow fragment) (KF) to the primer-templates containing a (+)-trans- or (+)-cis-B[a]P-N(2)-dG adduct. When the primer was terminated one nucleotide before the adduct, the presence of a (+)-trans-B[a]P-N(2)-dG adduct did not affect the binding while a (+)-cis-B[a]P-N(2)-dG adduct caused a slight decrease in affinity. The presence of any dNTP decreased the affinity of KF to the modified primer-templates. (In contrast, a strong increase of the affinity to unmodified primer-templates was observed in the presence of the next correct dNTP.) Limited protease digestion experiments indicated that a closed ternary complex of KF with the modified primer-templates was not detectable in the presence of any dNTP, whereas it was clearly observed with unmodified template in the presence of the next correct nucleotide. These findings suggest that these adducts may interfere with the conformational change to the catalytically active closed ternary complex and/or cause significant destabilization of this complex. When the primers extended to the position across from the adduct, the affinity of KF was significantly decreased irrespective of the identity of the base across from the adduct, possibly explaining the low bypass of the lesion. Interestingly, the stability of these DNA-polymerase complexes correlated with nucleotide insertion kinetics for the unmodified and (+)-trans-B[a]PDE-modified templates.




new topics/pols set partial results complete validated


No results available for this paper.

Entry validated by:

Using Polbase tables:


Tables may be sorted by clicking on any of the column titles. A second click reverses the sort order. <Ctrl> + click on the column titles to sort by more than one column (e.g. family then name).


It is also possible to filter the table by typing into the search box above the table. This will instantly hide lines from the table that do not contain your search text.