Fidelity Mechanisms of DNA Polymerase Beta

PROJECT SUMMARY/ABSTRACT Mutations in the human genome give rise to many human diseases including cancer. Mutations are also beneficial for processes including evolution and adaptive immunity. However, little is known about the molecular basis of mutagenesis. One mechanism that leads to the introduction of mutations is

misincorporation of nucleotides by DNA polymerases during DNA replication and repair. The focus of our application is on deoxynucleoside triphosphate (dNTP) substrate selection by DNA polymerase beta (Pol β). Pol β functions in base excision repair, a process that has evolved to repair oxidative DNA damage. These

types of damage are generated by a cell’s endogenous metabolism as well as exogenous sources such as ionizing radiation, UV light, and chemotherapeutic agents. During base excision repair, 20,000-50,000 predominantly oxidative DNA lesions are enzymatically removed, leaving small gaps in the DNA that are filled

in by Pol β. Incorrect substrate selection by Pol β during base excision repair has potential to result in genomic instability. We have recently shown that Pol β also functions in DNA gap filling during microhomology-mediated end-joining in VDJ recombination, where it plays an important role in generating immune diversity.

Our broad, long-term objective is to understand how DNA polymerases choose the correct substrate for incorporation into DNA. Pol β is an excellent model for studying polymerase mechanisms because of its relatively small size and the ease of purifying protein and growing crystals. The aims of this application are to

understand how Pol β selects the correct dNTP and why it sometimes chooses the incorrect dNTP for incorporation into DNA. We will employ a powerful combination of biochemical, structural, biophysical, computational and biological approaches in this multi-investigator project. We will determine how Pol β selects

the correct substrate for incorporation into DNA and how the mechanisms differ for mutator variants of Pol β. Our research will result in the characterization of the roles of amino acid residues in the selection of substrates during Pol β catalysis. Our findings will be of fundamental importance in understanding the molecular basis of

substrate selection by Pol β that will likely have broad applicability to other DNA polymerases.