Critical amino acids in Escherichia coli UmuC responsible for sugar discrimination and base-substitution fidelity

Nucleic Acids Res. 2012 Jul;40(13):6144-57. doi: 10.1093/nar/gks233. Epub 2012 Mar 15.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Base Pairing
  • Carbohydrates / chemistry
  • DNA / biosynthesis
  • DNA / chemistry
  • DNA Replication
  • DNA-Directed DNA Polymerase / chemistry*
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / metabolism*
  • Escherichia coli Proteins / chemistry*
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism*
  • Models, Molecular
  • Mutagenesis
  • Mutation
  • RNA / biosynthesis
  • Ribonucleotides / metabolism
  • Substrate Specificity


  • Carbohydrates
  • Escherichia coli Proteins
  • Ribonucleotides
  • RNA
  • DNA
  • UmuC protein, E coli
  • DNA polymerase V, E coli
  • DNA-Directed DNA Polymerase