title: Genetic requirement for mutagenesis of the G[8,5-Me]T cross-link in Escherichia coli: DNA polymerases IV and V compete for error-prone bypass.
abstract: γ-Radiation generates a variety of complex lesions in DNA, including the G[8,5-Me]T intrastrand cross-link in which C8 of guanine is covalently linked to the 5-methyl group of the 3'-thymine. We have investigated the toxicity and mutagenesis of this lesion by replicating a G[8,5-Me]T-modified plasmid in Escherichia coli with specific DNA polymerase knockouts. Viability was very low in a strain lacking pol II, pol IV, and pol V, the three SOS-inducible DNA polymerases, indicating that translesion synthesis is conducted primarily by these DNA polymerases. In the single-polymerase knockout strains, viability was the lowest in a pol V-deficient strain, which suggests that pol V is most efficient in bypassing this lesion. Most mutations were single-base substitutions or deletions, though a small population of mutants carrying two point mutations at or near the G[8,5-Me]T cross-link was also detected. Mutations in the progeny occurred at the cross-linked bases as well as at bases near the lesion site, but the mutational spectrum varied on the basis of the identity of the DNA polymerase that was knocked out. Mutation frequency was the lowest in a strain that lacked the three SOS DNA polymerases. We determined that pol V is required for most targeted G → T transversions, whereas pol IV is required for the targeted T deletions. Our results suggest that pol V and pol IV compete to carry out error-prone bypass of the G[8,5-Me]T cross-link.
authors.affiliation: Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269, United States.
authors.forename: Paromita
authors.initials: P
authors.lastname: Raychaudhury
authors.forename: Ashis K
authors.initials: AK
authors.lastname: Basu
mesh_terms: D001483:Base Sequence; D004249:DNA Damage / Q000235:genetics*; D019951:DNA Polymerase beta / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D055786:Gene Knockout Techniques; D050296:Microbial Viability / Q000235:genetics; D016296:Mutagenesis; D017384:Sequence Deletion
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D019951:DNA Polymerase beta; C120926:DNA polymerase V, E coli; D004259:DNA-Directed DNA Polymerase