The overall goal of this project is to understand the molecular mechanism of template switching by DNA polymerases involved in DNA repair and translesion synthesis. Template switching is the ability to synthesize an uninterrupted polynucleotide chain across two discontinuous template strands usually resulting from a strand break. It is seen in vitro in a number of eukaryotic, archeal and prokaryotic DNA polymerases. The in vivo significance of template switching by DNA polymerases remains unclear, although it might be involved in non-homologous end joining or during replication fork collapse at DNA double-strand breaks. There is also strong in vivo evidence to suggest that template switching occurs at damaged DNA sites and at sites containing quasipalindromic sequences (i.e. imperfect inverted repeats). This work addresses basic questions on the nature of DNA polymerases and how they interact with strand breaks. It seeks to understand the scope of this activity by examining template switching in several classes of human DNA polymerases in order to establish a general mechanism for all DNA polymerase. It also seeks to understand how significant this activity might be in generating mutations: the driving force of evolution. Previous NSF-supported work from the PI's laboratory showed that human DNA polymerase alpha, an important replicative DNA polymerase involved in lagging strand synthesis, exhibited template-switching activity. The research described here seeks to broaden the investigation by asking whether other human DNA polymerases can also perform template switching. In particular can the enzymes in the so-called X and Y family of DNA polymerases also perform template switching? The members of these two distinct DNA polymerase families are generally involved in DNA damage tolerance or repair. Focusing on biochemical experiments and using purified proteins and defined DNA substrates that mimic a DNA double-strand break, the experimental strategy is to test the hypothesis that these enzymes perform template switching in vitro. Enzymes involved in template switching will be further characterized by structure/function analysis to determine important domains on the protein required for activity. Candidate enzymes will also be characterized with respect to their binding affinity to various template-switching substrates in order to determine common structural motifs in the DNA substrates that may necessary for template switching.
This project will strengthen the PI's existing strategy to integrate undergraduate research participation from the classroom to the research laboratory in a coherent series of projects that students can pursue in upper-division molecular biology and biotechnology courses, leading seamlessly to research. The PI, himself a member of a group historically under-represented in the natural sciences, has also developed a program that encourages undergraduate research participation from under-represented minority groups.
