The E249K mutator mutant of DNA polymerase beta extends mispaired termini

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 m utation assay. The basis for this loss of accuracy was investigated by meas urement of misincorporation fidelity in single turnover conditions. For the four misincorporation reactions investigated, the fidelity of the E249K mu tant was not significantly different from wild type, implying that the muta tor 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 enzym e occurred less during the conformational change and chemical steps and mor e during the initial binding step, compared with pol beta wild type. This i mplies that the E249R mutation alters the kinetic mechanism of nucleotide d iscrimination without reducing misincorporation fidelity. In a missing base primer extension assay, we observed that the mutant enzyme produced mispai rs and extended them. This indicates that the altered fidelity of E249K cou ld 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 efficie ntly. These results demonstrate that an enhanced ability to extend mispairs can produce a mutator phenotype and that the Glu-249 side chain of DNA pol ymerase beta is critical for mispair extension fidelity.