A Collapsed Fingers Subdomain is the Basis for DNA Polymerase β I260M Mutator Activity

Abstract

DNA Polymerase β fills single nucleotide gaps as a part of the Base Excision Repair pathway (BER); thus, deficiencies in Pol β can lead to increased mutation frequency in the cell, which can result in cancer. Our lab has previously shown that the I260M germline mutation of Pol β, which was first identified in prostate cancer, has reduced nucleotide selectivity in a sequence context dependent manner, incorporating G opposite A. To identify the molecular mechanism of the reduced fidelity of I260M, we studied polymerization using single turnover kinetics, and conformational changes using steady state fluorescence and stopped flow FRET. Our data indicate that the I260M mutation affects the fingers region of Pol β by creating a “collapsed” state in both the opened (in the absence of nucleotide) and closed (prior to chemistry) states. Importantly, we show that the incorrect incoming nucleotide binds more tightly to I260M when compared to the wild‐type by a factor of 3.4x. Based on this data, we found that the collapsed fingers subdomain state of I260M may decrease nucleotide discrimination in I260M, illustrating the importance of the “fingers closing” conformational change for polymerase fidelity. A model is being developed to compare the rate of the “fingers closing” conformational change and the reverse reaction of nucleotide release between I260M and the WT.