Collapsed State\nMediates the Low Fidelity of the DNA\nPolymerase β I260 Mutant

DNA polymerase β (Pol β) fills single nucleotide\ngaps\nduring base excision repair. Deficiencies in Pol β can lead\nto increased mutagenesis and genomic instability in the cell, resulting\nin cancer. Our laboratory has previously shown that the I260 M somatic\nmutation of Pol β, which was first identified in prostate cancer,\nhas reduced nucleotide discrimination in a sequence context-dependent\nmanner. I260 M incorporates the incorrect G opposite A in this context\nmore readily than WT. To identify the molecular mechanism of the reduced\nfidelity of I260M, we studied incorporation using single turnover\nkinetics and the nature and rates of conformational changes using\nsteady-state fluorescence and Förster resonance energy transfer\n(FRET). Our data indicate that the I260 M mutation affects the fingers\nregion of rat Pol β by creating a “collapsed”\nstate in both the open (in the absence of nucleotide) and closed (prior\nto chemistry) states. I260 M is a temperature-sensitive mutator and\nbinds nucleotides tighter than the WT protein, resulting in reduced\nfidelity compared to the WT. Additionally, we have generated a kinetic\nmodel of WT and I260 M using FRET and single turnover data, which\ndemonstrates that I260 M precatalytic conformation changes differ\ncompared to the WT as it is missing a precatalytic noncovalent step.\nTaken together, these results suggest that the collapsed state of\nI260 M may decrease its ability for nucleotide discrimination, illustrating\nthe importance of the “fingers closing” conformational\nchange for polymerase fidelity and accurate DNA synthesis.