A COLLAPSED FINGERS SUBDOMAIN IS THE BASIS FOR DNA POLYMERASE Β I260M MUTATOR ACTIVITY

Abstract

DNA Polymerase β (Pol 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 genomic instability and cancer. Our lab has previously shown that the I260M somatic mutation of Pol β, which was first identified in prostate cancer, has reduced nucleotide discrimination in a sequence context-dependent manner. I260M incorporates the incorrect G opposite A in this context more readily than WT. To identify the molecular mechanism of the reduced fidelity of I260M, we studied incorporation using single turnover kinetics, and conformational changes using steady-state fluorescence and stopped-flow FRET. Our data indicates that the I260M mutation affects the fingers region of Pol β by creating a “collapsed” state in both the open (in the absence of nucleotide) and closed (prior to chemistry) states. Additionally, using the same technique, we visualized fingers movement during incorporation of an incorrect nucleotide for the first time. I260M binds these nucleotides tighter than the wildtype does and has 3.4 times reduced fidelity compared to the WT, explaining its mutator phenotype. Taken together, these results suggest that the collapsed state of I260M may decrease its ability for nucleotide discrimination, illustrating the importance of the “fingers closing” conformational change for polymerase fidelity. Additionally, we have built a kinetic model of WT and I260M using stopped-flow FRET and single turnover data, which demonstrates that I260M is also missing a non-covalent step (NCS).