Novel function and regulation of mutagenic DNA polymerases in Escherichia coli
Author(s)Jarosz, Daniel F
Massachusetts Institute of Technology. Dept. of Chemistry.
Graham C. Walker.
MetadataShow full item record
The observation that mutations in the Escherichia coli genes umuC+ and umuD+ abolish mutagenesis induced by UV-light strongly supported the counterintuitive notion that such mutagenesis is an active rather than passive process. Biochemical studies have revealed that umuC+ and its homolog dinB+ encode novel, low to moderate fidelity DNA polymerases with the ability to catalyze synthesis on imperfect DNA templates in a process termed translesion synthesis (TLS). Similar enzymes exist in nearly all organisms, constituting the Y-superfamily of DNA polymerases. Although DinB is the only Y-family DNA polymerase conserved among all domains of life, its precise function has remained elusive. Here we show that AdinB E. coli strains are sensitive to DNA damaging agents that form lesions at the N2 position of guanine. In vitro bypass studies of an N2-guanine adduct by DinB demonstrate considerable preference for correct nucleotide insertion and an increased catalytic proficiency on the lesion-bearing template relative to undamaged DNA. Moreover, DinB and its mammalian and archaeal orthologs possess similar substrate specificities. Mutation of a single residue in the active site ofE. coli DinB suggests that its enhanced activity is coupled to lesion recognition and that its TLS function is required for resistance to DNA damaging agents in vivo.(cont.) Regulation of the mutagenic potential of DinB is critical for maintenance of genomic integrity. We present evidence indicating that abortive TLS products generated by a DinB variant are subject to the proofreading function of DNA polymerase III. Moreover, both the TLS activity and -1 frameshift mutator potential of DinB are modulated in a highly sophisticated manner by the DNA damage-inducible proteins RecA and UmuD2. These biochemical data, coupled with genetic analyses and molecular modeling, indicate that DinB is a specialized and remarkably controlled translesion DNA polymerase. In addition, we present evidence that the umuC+participates in several novel biological functions in addition to its established role in TLS. A novel umuC gain-of-function allele confers striking resistance to hydroxyurea and umuC+ mediates the expression of genes and physiological responses under conditions of SOS induction. Taken together, these observations hint at at a largely uncharacterized function of Y-family polymerases in sculpting physiological responses, including active mechanisms of cell death, in response to environmental stress.
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2007.Vita.Includes bibliographical references.
DepartmentMassachusetts Institute of Technology. Dept. of Chemistry.
Massachusetts Institute of Technology