Elucidation of kinetic mechanisms of human translesion DNA polymerase κ using tryptophan mutants

FEBS J. 2014 Oct;281(19):4394-410. doi: 10.1111/febs.12947. Epub 2014 Aug 14.

Abstract

To investigate the conformational dynamics of human DNA polymerase κ (hpol κ), we generated two mutants, Y50W (N-clasp region) and Y408W (linker between the thumb and little finger domains), using a Trp-null mutant (W214Y/W392H) of the hpol κ catalytic core enzyme. These mutants retained catalytic activity and similar patterns of selectivity for bypassing the DNA adduct 7,8-dihydro-8-oxo-2'-deoxyguanosine, as indicated by the results of steady-state and pre-steady-state kinetic experiments. Stopped-flow kinetic assays with hpol κ Y50W and T408W revealed a decrease in Trp fluorescence with the template G:dCTP pair but not for any mispairs. This decrease in fluorescence was not rate-limiting and is considered to be related to a conformational change necessary for correct nucleotidyl transfer. When a free 3'-hydroxyl was present on the primer, the Trp fluorescence returned to the baseline level at a rate similar to the observed kcat , suggesting that this change occurs during or after nucleotidyl transfer. However, polymerization rates (kpol ) of extended-product formation were fast, indicating that the slow fluorescence step follows phosphodiester bond formation and is rate-limiting. Pyrophosphate formation and release were fast and are likely to precede the slower relaxation step. The available kinetic data were used to fit a simplified minimal model. The extracted rate constants confirmed that the conformational change after phosphodiester bond formation was rate-limiting for hpol κ catalysis with the template G:dCTP pair.

Keywords: DNA polymerase; DNA replication; conformational change; enzyme kinetics; translesion synthesis.

Publication types

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

MeSH terms

  • Amino Acid Substitution
  • Catalytic Domain
  • DNA Adducts / chemistry
  • DNA-Directed DNA Polymerase / chemistry*
  • DNA-Directed DNA Polymerase / genetics
  • Deoxycytosine Nucleotides / chemistry
  • Diphosphates / chemistry
  • Humans
  • Kinetics
  • Mutagenesis, Site-Directed
  • Protein Binding

Substances

  • DNA Adducts
  • Deoxycytosine Nucleotides
  • Diphosphates
  • 2'-deoxycytidine 5'-triphosphate
  • diphosphoric acid
  • DNA-Directed DNA Polymerase
  • POLK protein, human