title: UmuD(2) inhibits a non-covalent step during DinB-mediated template slippage on homopolymeric nucleotide runs.
abstract: Escherichia coli DinB (DNA polymerase IV) possesses an enzyme architecture resulting in specialized lesion bypass function and the potential for creating -1 frameshifts in homopolymeric nucleotide runs. We have previously shown that the mutagenic potential of DinB is regulated by the DNA damage response protein UmuD(2). In the current study, we employ a pre-steady-state fluorescence approach to gain a mechanistic understanding of DinB regulation by UmuD(2). Our results suggest that DinB, like its mammalian and archaeal orthologs, uses a template slippage mechanism to create single base deletions on homopolymeric runs. With 2-aminopurine as a fluorescent reporter in the DNA substrate, the template slippage reaction results in a prechemistry fluorescence change that is inhibited by UmuD(2). We propose a model in which DNA templates containing homopolymeric nucleotide runs, when bound to DinB, are in an equilibrium between non-slipped and slipped conformations. UmuD(2), when bound to DinB, displaces the equilibrium in favor of the non-slipped conformation, thereby preventing frameshifting and potentially enhancing DinB activity on non-slipped substrates.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: James J
authors.initials: JJ
authors.lastname: Foti
authors.forename: Angela M
authors.initials: AM
authors.lastname: Delucia
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Joyce
authors.forename: Graham C
authors.initials: GC
authors.lastname: Walker
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage; D019951:DNA Polymerase beta / Q000235:genetics / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011938:Rec A Recombinases / Q000378:metabolism; D012697:Serine Endopeptidases / Q000494:pharmacology*; D013698:Templates, Genetic
chemical_list: C094399:DinB protein, E coli; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D009711:Nucleotides; D004247:DNA; D011938:Rec A Recombinases; D019951:DNA Polymerase beta; D004259:DNA-Directed DNA Polymerase; C055218:UmuD protein, E coli; D012697:Serine Endopeptidases
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