title: Critical amino acids in Escherichia coli UmuC responsible for sugar discrimination and base-substitution fidelity.
abstract: The active form of Escherichia coli DNA polymerase V responsible for damage-induced mutagenesis is a multiprotein complex (UmuD'(2)C-RecA-ATP), called pol V Mut. Optimal activity of pol V Mut in vitro is observed on an SSB-coated single-stranded circular DNA template in the presence of the β/γ complex and a transactivated RecA nucleoprotein filament, RecA*. Remarkably, under these conditions, wild-type pol V Mut efficiently incorporates ribonucleotides into DNA. A Y11A substitution in the 'steric gate' of UmuC further reduces pol V sugar selectivity and converts pol V Mut into a primer-dependent RNA polymerase that is capable of synthesizing long RNAs with a processivity comparable to that of DNA synthesis. Despite such properties, Y11A only promotes low levels of spontaneous mutagenesis in vivo. While the Y11F substitution has a minimal effect on sugar selectivity, it results in an increase in spontaneous mutagenesis. In comparison, an F10L substitution increases sugar selectivity and the overall fidelity of pol V Mut. Molecular modeling analysis reveals that the branched side-chain of L10 impinges on the benzene ring of Y11 so as to constrict its movement and as a consequence, firmly closes the steric gate, which in wild-type enzyme fails to guard against ribonucleoside triphosphates incorporation with sufficient stringency.
authors.affiliation: Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-3371, USA.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Vaisman
authors.forename: Wojciech
authors.initials: W
authors.lastname: Kuban
authors.forename: John P
authors.initials: JP
authors.lastname: McDonald
authors.forename: Kiyonobu
authors.initials: K
authors.lastname: Karata
authors.forename: Wei
authors.initials: W
authors.lastname: Yang
authors.forename: Myron F
authors.initials: MF
authors.lastname: Goodman
authors.forename: Roger
authors.initials: R
authors.lastname: Woodgate
mesh_terms: D019943:Amino Acid Substitution; D020029:Base Pairing; D002241:Carbohydrates / Q000737:chemistry; D004247:DNA / Q000096:biosynthesis / Q000737:chemistry; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D012313:RNA / Q000096:biosynthesis; D012265:Ribonucleotides / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D002241:Carbohydrates; D029968:Escherichia coli Proteins; D012265:Ribonucleotides; D012313:RNA; D004247:DNA; C078202:UmuC protein, E coli; C120926:DNA polymerase V, E coli; D004259:DNA-Directed DNA Polymerase