Sequence and functional analyses of DNA polymerase A enzymes from bacteriophages to yeast

DNA polymerases are essential enzymes required to accurately and rapidly copy genetic material. Although they conserve a common function of duplicating DNA, DNA polymerases are incredibly diverse enzymes. The polymerase A (PolA) family of DNA polymerases perform a wide range of functions in diverse systems ranging from viruses to eukaryotic cells. PolAs at a minimum contain a polymerase domain that synthesizes DNA and a 3’-5’ exonuclease domain that remove mistakes made during replication. PolAs have evolved to obtain additional protein domains that allow them to perform functions ranging from synthesizing short stretches of DNA during DNA repair to being the replicative polymerase tasked with duplicating the entire genome. The goals of this work are to compare PolA enzymes from both bacteriophages and fungi to better understand how these enzymes use additional protein domains for specialized function. Bacteriophages are viruses that only infect bacteria, and due to recent work by undergraduate students that are part of HHMI’s Science Education Alliance, there are now 3,731 fully sequenced Actinobacteriophage genomes. Analysis of these genomes have revealed that many, but not all, of these phages contain a PolA enzyme, and we have become interested in the function of these enzymes in phage replication and repair. Our analysis identified 1,351 PolA sequences in viruses that infect several different bacterial hosts, and these proteins range in size from as little as 582 to as large as 801 residues. These enzymes appear to conserve essential catalytic residues needed for polymerase and exonuclease activities, and the variation in protein sizes are due to additional novel domains present in these polymerases. These domains include glycosylase domains, which implicate these enzymes in DNA repair. The goals of this work are to compare these enzymes to the well-studied PolA from bacteriophage T7 to better understand how PolA enzymes have evolved in bacteriophages. Another member of the PolA family is DNA polymerase gamma (PolG), which is a replicative polymerase found in the mitochondria. The second part of this work focuses on the PolG enzyme from the pathogenic fungus Cryptococcus neoformans, which causes cryptococcosis of the lungs and central nervous system. The C. neoformans PolG enzyme has yet to be biochemically studied, and we have identified two novel domains that are not present in other PolGs. Due to their location and amino acid content, we predict that these domains either bind DNA or to partner proteins during mitochondrial DNA replication. The goals of this work are to perform extensive bioinformatics analysis of the novel domains to learn more about possible function and to confirm whether these novel domains bind partner proteins. The N-terminal domain has high sequence conservation and is only found in Cryptococcus species, while the internal domain, located between the spacer and the polymerase domains, lacks residue conservation and is found in a variety of species. We have successfully expressed and purified both the full-length enzyme as well as just the N-terminal domain, and pull-down assays were optimized to identify cryptococcal proteins that bind to PolG