title: Elucidation of kinetic mechanisms of human translesion DNA polymerase κ using tryptophan mutants.
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.
authors: Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN, USA; Department of Chemistry and Biochemistry, Central Michigan University, Mount Pleasant, MI, USA.
authors: Linlin
authors: L
authors: Zhao
authors: Matthew G
authors: MG
authors: Pence
authors: Robert L
authors: RL
authors: Eoff
authors: Shuai
authors: S
authors: Yuan
authors: Catinca A
authors: CA
authors: Fercu
authors: F Peter
authors: FP
authors: Guengerich
mesh_terms: D019943:Amino Acid Substitution; D020134:Catalytic Domain; D018736:DNA Adducts / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics; D003845:Deoxycytosine Nucleotides / Q000737:chemistry; D011756:Diphosphates / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D000099270:Y-Family DNA Polymerases
chemical_list: D018736:DNA Adducts; D004259:DNA-Directed DNA Polymerase; D003845:Deoxycytosine Nucleotides; D011756:Diphosphates; C024107:2'-deoxycytidine 5'-triphosphate; C107241:diphosphoric acid; C404302:POLK protein, human; D000099270:Y-Family DNA Polymerases
keywords: DNA polymerase; DNA replication; conformational change; enzyme kinetics; translesion synthesis