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J Biol Chem. 2015 Feb 6;290(6):3825-35. doi: 10.1074/jbc.M114.615278. Epub 2014 Dec 23.

Yeast DNA polymerase ϵ catalytic core and holoenzyme have comparable catalytic rates.

Author information

1
From the Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden.
2
From the Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden erik.johansson@medchem.umu.se.

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.

KEYWORDS:

DNA Polymerase; DNA Repair; DNA Replication; Enzyme Catalysis; Enzyme Kinetics

PMID:
25538242
PMCID:
PMC4319046
DOI:
10.1074/jbc.M114.615278
[Indexed for MEDLINE]
Free PMC Article

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