期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 290, 期 6, 页码 3825-3835出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M114.615278
关键词
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资金
- Swedish Research Council
- Swedish Cancer Society
- Kempe Foundations
- Knut and Alice Wallenberg Foundation
- Insamlingstiftelsen at the medical faculty of Umea Universitet
The holoenzyme of yeast DNA polymerase epsilon (Pol epsilon) consists of four subunits: Pol2, Dpb2, Dpb3, and Dpb4. A protease-sensitive site results in an N-terminal proteolytic fragment of Pol2, called Pol2(core), that consists of the catalytic core of Pol epsilon 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 epsilon and Pol2(core). Single-turnover pre-steady- state kinetics also showed that the k(pol) of Pol epsilon and Pol2(core) were comparable when preloading the polymerase onto the primer-template before adding Mg2+ epsilon 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 epsilon than for Pol2(core). The largest difference between Pol epsilon and Pol2(core) was observed when challenged for the formation of a ternary complex and incorporation of the first nucleotide. Pol epsilon needed less than 1 s to incorporate a nucleotide, but several seconds passed before Pol2(core) 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 epsilon but facilitate the loading and incorporation of the first nucleotide by Pol epsilon.
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