title: Yeast DNA polymerase ϵ catalytic core and holoenzyme have comparable catalytic rates.
abstract: The holoenzyme of yeast DNA polymerase ϵ (Pol ϵ) consists of four subunits: Pol2, Dpb2, Dpb3, and Dpb4. A protease-sensitive site results in an N-terminal proteolytic fragment of Pol2, called Pol2core, that consists of the catalytic core of Pol ϵ and retains both polymerase and exonuclease activities. Pre-steady-state kinetics showed that the exonuclease rates on single-stranded, double-stranded, and mismatched DNA were comparable between Pol ϵ and Pol2core. Single-turnover pre-steady-state kinetics also showed that the kpol of Pol ϵ and Pol2core were comparable when preloading the polymerase onto the primer-template before adding Mg(2+) and dTTP. However, a global fit of the data over six sequential nucleotide incorporations revealed that the overall polymerization rate and processivity were higher for Pol ϵ than for Pol2core. The largest difference between Pol ϵ and Pol2core was observed when challenged for the formation of a ternary complex and incorporation of the first nucleotide. Pol ϵ needed less than 1 s to incorporate a nucleotide, but several seconds passed before Pol2core incorporated detectable levels of the first nucleotide. We conclude that the accessory subunits and the C terminus of Pol2 do not influence the catalytic rate of Pol ϵ but facilitate the loading and incorporation of the first nucleotide by Pol ϵ.
authors.affiliation: From the Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden.
authors.forename: Rais A
authors.initials: RA
authors.lastname: Ganai
authors.affiliation: From the Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden.
authors.forename: Pia
authors.initials: P
authors.lastname: Osterman
authors.affiliation: From the Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden erik.johansson@medchem.umu.se.
authors.forename: Erik
authors.initials: E
authors.lastname: Johansson
mesh_terms: D004257:DNA Polymerase II / Q000378:metabolism*; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism; D020035:Holoenzymes / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013942:Thymine Nucleotides / Q000378:metabolism
chemical_list: D004277:DNA, Single-Stranded; D020035:Holoenzymes; D029701:Saccharomyces cerevisiae Proteins; D013942:Thymine Nucleotides; D004257:DNA Polymerase II; D008274:Magnesium; C024157:thymidine 5'-triphosphate
keywords: DNA Polymerase; DNA Repair; DNA Replication; Enzyme Catalysis; Enzyme Kinetics