Modifying the β,γ Leaving-Group Bridging Oxygen Alters Nucleotide Incorporation Efficiency, Fidelity, and the Catalytic Mechanism of DNA Polymerase β

• Published in: Biochemistry (Easton) 2007-01, Vol.46 (2), p.461-471
• Main Authors: Sucato, Christopher A, Upton, Thomas G, Kashemirov, Boris A, Batra, Vinod K, Martínek, Václav, Xiang, Yun, Beard, William A, Pedersen, Lars C, Wilson, Samuel H, McKenna, Charles E, Florián, Jan, Warshel, Arieh, Goodman, Myron F
• Format: Article
• Language: English
• Subjects: Base Sequence; Catalytic Domain; Crystallography, X-Ray; Deoxyguanine Nucleotides - chemistry; Deoxyguanine Nucleotides - metabolism; DNA - biosynthesis; DNA - chemistry; DNA - genetics; DNA Polymerase beta - chemistry; DNA Polymerase beta - genetics; DNA Polymerase beta - metabolism; Humans; In Vitro Techniques; Kinetics; Models, Molecular; Nucleic Acid Conformation; Nucleotides - chemistry; Nucleotides - metabolism; Oxygen - metabolism; Protein Conformation; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Substrate Specificity; Thermodynamics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi061517b

DNA polymerase catalysis and fidelity studies typically compare incorporation of “right” versus “wrong” nucleotide bases where the leaving group is pyrophosphate. Here we use dGTP analogues replacing the β,γ-bridging O with CH2, CHF, CF2, or CCl2 to explore leaving-group effects on the nucleotidyl transfer mechanism and fidelity of DNA polymerase (pol) β. T·G mismatches occur with fidelities similar to dGTP with the exception of the CH2 analogue, which is incorporated with 5-fold higher fidelity. All analogues are observed to bind opposite template C with K ds between 1 and 4 μM, and structural evidence suggests that the analogues bind in essentially the native conformation, making them suitable substrates for probing linear free energy relationships (LFERs) in transient-kinetics experiments. Importantly, Brønsted correlations of log(k pol) versus leaving-group pK a for both right and wrong base incorporation reveal similar sensitivities (βlg ≈ −0.8) followed by departures from linearity, suggesting that a chemical step rather than enzyme conformational change is rate-limiting for either process. The location of the breaks relative to pK as of CF2, O, and the sterically bulky CCl2-bridging compounds suggests a modification-induced change in the mechanism by stabilization of leaving-group elimination. The results are addressed theoretically in terms of the energetics of successive primer 3‘-O addition (bond forming) and pyrophosphate analogue elimination (bond breaking) reaction energy barriers.