A DNA polymerase beta mutator mutant with reduced nucleotide discrimination and increased protein stability

DNA polymerase beta (pol beta) offers a simple system to examine the role o f polymerase structure in the fidelity of DNA synthesis. In this study, the M282L variant of pol beta (M282L beta) was identified using an in vivo gen etic screen. Met282, which does not contact the DNA template or the incomin g deoxynucleoside triphosphate (dNTP) substrate, is located on alpha -helix N of pol beta. This mutant enzyme demonstrates increased mutagenesis in bo th in vivo and in vitro assays. M282L beta has a 7.5-fold higher mutation f requency than wild-type pol beta; M282L beta commits a variety of base subs titution and frameshift errors. Transient-state kinetic methods were used t o investigate the mechanism of intrinsic mutator activity of M282L. Results show an 11-fold decrease in dNTP substrate discrimination at the level of groundstate binding. However, during the protein conformational change and/ or phosphodiester bond formation, the nucleotide discrimination is improved . X-ray crystallography was utilized to gain insights into the structural b asis of the decreased DNA synthesis fidelity. Most of the Structural change s are localized to site 282 and the surrounding region in the C-terminal pa rt of the 31-k-Da domain. Repositioning of mostly hydrophobic amino acid re sidues in the core of the C-terminal portion generates a protein with enhan ced stability. The combination of structural and equilibrium unfolding data suggests that the mechanism of nucleotide discrimination is possibly affec ted by the compacting of the hydrophobic core around residue Leu282. Subseq uent movement of an adjacent surface residue, Arg283, produces a slight inc rease in volume of the pocket that may accommodate the incoming correct bas e pair. The structural changes of M282L beta ultimately lead to an overall reduction in polymerase fidelity.