Determination of Escherichia coli Genes Important for DNA Repair Following Alkylation

Abstract

Alkylating agents, common in industrial manufacturing and chemotherapy, can damage DNA by adding alkyl groups to nucleotides. Alkylation can decrease the stability of the N‐glycosidic sugar‐base bond, making it more apt to break; alkylation can also interfere with base pairing. Either of these perturbations can lead to mutations if not remedied. DNA repair mechanisms, including direct base repair, base excision repair, and nucleotide excision repair, play important roles in correcting these lesions. Our research aims to better understand the mechanisms behind repair of alkylated DNA, first by confirming which genes are most critical. An assay was designed to screen bacterial strains harboring single deletions of more than 20 genes, selected based on prior known association to DNA repair. The survival of E. coli AB1157 strains, each with one of the 20 genes deleted, was measured upon exposure to one of two alkylating agents: styrene oxide (SO) or chloroacetaldehyde (CAA). Strains with dam deleted were sensitive to SO and CAA, suggesting that DNA adenine methylase, the protein encoded by dam, plays a critical role in DNA alkylation repair. Strains lacking recA, which plays multiple roles in DNA damage responses, were quite sensitive to both agents. Strains with mfd, mutH, or mutY deleted were not more sensitive to SO and CAA than the wild‐type strains. Next, the survival of double knockouts will be assayed, which will help determine if these genes act in the same or different DNA repair pathways.Support or Funding InformationThis work was supported by the American Cancer Society and the NU Office of the Provost.