Abstract
DNA is a molecular information carrier that is essential to life. DNA Polymerase III is a complex that catalyzes DNA replication, maintaining genomic integrity that is needed for Escherichia coli survival. The core of the replisome contains DnaQ, a 3’-5’ exonuclease that proofreads newly synthesized DNA. YoaA is a 5’-3’ accessory helicase that permits the excision of bases on nascent DNA. The Lovett Lab discovered that YoaA forms a physical interaction with DnaQ, which is compelling because both are key to DNA repair. This study seeks to characterize this interaction. I biochemically pulled down 6X-His-tagged DnaQ using Biotin-Binding Domain-tagged YoaA as bait. This successful pulldown is supplemental to existing evidence of the YoaA-DnaQ interaction. An AlphaFold III model predicted residues on YoaA for affinity to DnaQ: T489, L490, and P491 were mutated to alanines to eliminate a potential interaction. These mutants were tested for YoaA’s AZT-tolerant phenotype. It was found that L490A and P491A are recessive loss-of-function alleles. Yeast two-hybrid analysis found that yoaA L490A breaks the YoaA-DnaQ interaction. I also found, using the cc105 system, that yoaA L490A might eliminate yoaA’s proofreading phenotype, though no Mann-Whitney significance was found. I suggest that yoaA L490A is an interaction-breaking mutant, though more work is needed to explore the phenotype of yoaA L490A. A cursory set of experiments searched for types of DNA damage that yoaA is sensitive to. yoaAΔ is not sensitive to phleomycin, hydroxyurea, mitomycin C, hydrogen peroxide, or t-butyl hydroperoxide, suggesting that YoaA might specifically permit excision of nucleosides.