Abstract
The DNA polymerase beta mutant enzyme, which is altered from glutamic acid to lysine at position 249, exhibits a mutator phenotype in primer extension assays and in the herpes simplex virus-thymidine kinase (HSV-tk) forward mutation assay. The basis for this loss of accuracy was investigated by measurement of misincorporation fidelity in single turnover conditions. For the four misincorporation reactions investigated, the fidelity of the E249K mutant was not significantly different from wild type, implying that the mutator phenotype was not caused by a general inability to distinguish between correct and incorrect bases during the incorporation reaction. However, the discrimination between correct and incorrect substrates by the E249K enzyme occurred less during the conformational change and chemical steps and more during the initial binding step, compared with pol beta wild type. This implies that the E249K mutation alters the kinetic mechanism of nucleotide discrimination without reducing misincorporation fidelity. In a missing base primer extension assay, we observed that the mutant enzyme produced mispairs and extended them. This indicates that the altered fidelity of E249K could be due to loss of discrimination against mispaired primer termini. This was supported by the finding that the E249K enzyme extended a G:A mispair 8-fold more efficiently than wild type and a C:T mispair 4-fold more efficiently. These results demonstrate that an enhanced ability to extend mispairs can produce a mutator phenotype and that the Glu-249 side chain of DNA polymerase beta is critical for mispair extension fidelity.
Highlights
DNA polymerase 1 is a mammalian polymerase that fills short gaps in DNA [1, 2]. pol  has been implicated in base excision repair [3, 4] and appears to function during meiosis [5]
In order to investigate the accuracy of the AZT-resistant pol  mutant enzymes, the ability of -WT to misincorporate and extend incorrect bases in a missing base assay was compared with the E249K, R253M, and D246V mutants
E249K was more effective than -WT in all four incomplete reactions, but the difference was most pronounced in the AϪ and CϪ reactions, representing misincorporation opposite T and G. pol  E249K, unlike -WT, was able to extend the primer past multiple misinsertion sites, indicating that the mutant enzyme is more willing to extend mispaired termini
Summary
Bacterial Strains—The strain BL21 DE3 was used for protein expression and has genotype FϪ ompT hsdSB(rBϪmBϪ) gal dcm (DE3). Single Turnover Misincorporation Assays—To elucidate the relative ability of the E249K enzyme compared with -WT to incorporate correct and incorrect dNTPs into a primer-template, we determined the equilibrium dissociation constant for dNTP binding, Kd, and the maximum rate of polymerization, kpol, for correct and incorrect dNTPs for each enzyme. Reactions were conducted at 37 °C in 50 mM Tris, pH 8.0, 10 mM MgCl2, 20 mM NaCl, 2 mM DTT, 0.2 mg/ml BSA, 2.5% glycerol, 50 nM 32P-end-labeled primer-template, and 750 nM enzyme. The data from the single turnover experiments were fit with a single exponential equation: [product] ϭ A(1 Ϫ exp(Ϫkobst)), where A is the amplitude and kobs is the observed first order rate constant for dNTP incorporation. Single Turnover Mispair Extension Assays—These assays were performed as described above, in single turnover conditions, except the primer-template contained mispaired termini
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