Insights into the Conformation of Aminofluorene-Deoxyguanine Adduct in a DNA Polymerase Active Site

Abstract

Background: DNA lesions in different sequence contexts exhibit alternate conformations. Results: DNA adduct conformation is differentially modulated by DNA structure, DNA polymerase, and the presence of an incoming nucleoside triphosphate. Conclusion: Adduct conformation is influenced by binding of a non-hydrolysable nucleotide to pol ␤ but not Klenow fragment. Significance: Adduct conformation and coding potential is modulated by constraints imposed by the polymerase active site.

Real-Time Surface Plasmon Resonance Study of Biomolecular Interactions between Polymerase and Bulky Mutagenic DNA Lesions

“…We previously observed similar conformational heterogeneity caused by FAF bound to 1 nt gap DNA in both the absence and presence of pol β. 7 The results are also consistent with translesion synthesis studies in which the minor groove conformation benzo[ a ]pyrene diol epoxide– N 2 -dG adducts creates steric clash with the active site of pol β, thereby reducing the insertion rate. 64 These results are in agreement with the steady-state kinetics data that show significant reductions in the f ins of dCTP opposite FABP in the CG*A and TG*A sequences (142- and 59-fold, respectively), relative to that of the corresponding unmodified controls.…”

“…We have recently reported a similar set of binding results for the N -deacetylated FAF. 7 With the correct nucleotide dCTP, the pol β binds 2.7-fold more tightly in the ternary complex than that in the binary complex and ∼3000-fold more tightly than to that of nongapped DNA. The binding affinity to the incorrect nucleotide was 4- to 5-fold lower than that to the correct dCTP.…”

“…The 32 P-labeled primer (50 pmol) was annealed with either an unmodified or adducted template oligonucleotide (60 pmol) by heating at 95 °C for 5 min and then slowly cooling to room temperature in 3 h. For pol β assays, a 1 nt gap was generated by adding a downstream 9-mer primer with 5′-phosphate group while annealing with radiolabeled primer (9-mer) and template (19-mer). 7 The ds/ss primer–template sequence (20 nM) was incubated with Kf-exo – (0.5 or 1.0 nM) for 5 min to form a binary complex in Tris buffer (Tris, 50 mM, pH 7.4; BSA, 50 μg/mL; 5% (v/v) glycerol). The reaction was initiated by adding a dNTP (100 μM)/MgCl 2 (5 mM) solution to a binary mixture and was incubated at 22 °C for 10 min.…”

“… 6 , 49 Subsequently, a similar study was conducted to elucidate how FAF lesions affect the active site conformation of the human repair enzyme pol β and how the structure and sequence of the DNA affects its ability to be repaired. 7 …”

“…As a result, the choice of polymerases used in the present study was based largely on the experimental systems in our previous work. 7 , 8 , 21 Obviously, future SPR studies should be expanded to a range of Y-family bypass polymerases, which are more likely to be involved in replication of bulky DNA lesions.…”