DNA Polymerase β:  Pre-Steady-State Kinetic Analysis and Roles of Arginine-283 in Catalysis and Fidelity

Abstract

DNA polymerase beta (pol beta) is the smallest and least complex DNA polymerase. The structure of the enzyme is well understood, but little is known about its catalytic properties, particularly processivity and fidelity. Pre-steady-state analysis of the incorporation of a single nucleotide into a short 25/45 oligonucleotide primer-template by pol beta was used to define the kinetic parameters of the polymerase. In addition, nucleotide analogs and site-specific mutants, along with structural analyses, were used to probe the structure-function relationship of pol beta. Several significant findings have been obtained: (i) The catalysis by pol beta is processive and displays an initial burst under pre-steady-state conditions, but the processivity is poor compared to other polymerases. (ii) The fidelity of pol beta is also low relative to other polymerases. (iii) Under pre-steady-state conditions the chemical step appears to be only partially rate-limiting on the basis of the low thio effect (4.3), defined as kpol(dNTP)/kpol(dNTP alpha S). The thio effect increases to 9 for incorporation of an incorrect nucleotide. These results are consistent with the existence of a substrate-induced conformational change that is also partially rate-limiting. (iv) A comparison between the two-dimensional NMR spectra of the wild-type and mutant enzymes indicates that the mutations at position 283 did not significantly perturb the structure of the enzyme. The conformational stability of the mutants is also unperturbed. Thus, R283 is not important to the overall structure of the enzyme. (v) The results of kinetic analyses of R283A and R283K mutants indicate that the hydrogen bond between R283 of pol beta and the template is important for catalysis. Both R283A and R283K mutants displayed decreases in catalytic efficiency by a factor of ca. 200 relative to wild-type pol beta. The mutants are also less faithful by a factor of 2-4, in terms of the T-G mispair vs the T-A correct pair. The perturbation, however, could occur at both the implied conformational step and the chemical step, since the thio effects of the mutants for both correct and incorrect nucleotides are similar to those of WT pol beta.

Orchestration of cooperative events in DNA synthesis and repair mechanism unraveled by transition path sampling of DNA polymerase β's closing

“…4) suggest that the rate-limiting step is associated with TS 3, namely the partial rotation of Arg-258 (overall barrier of 20.5 Ϯ 3 k B T). This value is lower but comparable to the overall rate of the reaction (25-28 k B T), as obtained from experimental kinetic data [k pol , the overall rate of nucleotide incorporation of 3-90 s Ϫ1 (27)(28)(29)(30)(31)(32)(33)]. This rate-limiting conformational barrier in the same order of the overall (conformational and chemical steps) reaction profile immediately suggests site-specific mutant experiments on Arg-258 to assess Arg-258's effect on reaction efficiency and fidelity.…”

“…1), pol ␤ fills single-stranded gaps in DNA with relatively high accuracy (fidelity): it recruits the nucleotide unit (dNTP, 2Ј-deoxyribonucleoside 5Ј-triphosphate) complementary to the template base (e.g., C opposite G) about 1,000 times more often than the incorrect unit (e.g., T opposite G) (27)(28)(29)(30)(31)(32)(33). Closed and open conformations of pol ␤ have been solved by x-ray crystallography by Wilson and coworkers (19,34) and are related by a large subdomain motion (Ϸ6 Å) of the thumb (Fig.…”

Base Substitution Specificity of DNA Polymerase β Depends on Interactions in the DNA Minor Groove

“…This side chain is hydrogen-bonded to the sugar of the preceding template nucleotide in the template⅐primer duplex. Previous studies have shown that the R283A mutant of pol ␤ has greatly reduced fidelity consistent with the importance of these interactions for polymerase selectivity (16,21,22). This phenotype includes an error rate of 25% for T⅐dGMP (16), suggesting complete loss of discrimination.…”

Threonine 79 Is a Hinge Residue That Governs the Fidelity of DNA Polymerase β by Helping to Position the DNA within the Active Site

“…Amino acid residues important for catalysis and fidelity, including Arg-283, Met-282, and Phe-272, are not poised for catalysis in the open conformation of the enzyme. However, once the enzyme assumes the closed conformation, these residues are brought into proper geometry for catalysis and for monitoring the fidelity of the chemical step (29,43). Residues Arg-283 and Phe-272 form hydrogen bonds and van der Waals contacts with the templating nucleotide in the closed conformation.…”