Yeast DNA Polymerase epsilon Catalytic Core and Holoenzyme Have Comparable Catalytic Rates
2014 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 6, 3825-3835 p.Article in journal (Refereed) Published
The holoenzyme of yeast DNApolymerase ε (Pol ε) consists of four subunits– Pol2, Dpb2, Dpb3, and Dpb4. A proteasesensitivesite results in a N-terminalproteolytic fragment of Pol2, called Pol2core,that consists of the catalytic core of Pol ε andretains both polymerase and exonucleaseactivities. Pre-steady-state kinetics showedthat the exonuclease rates on single-stranded,double-stranded, and mismatched DNA werecomparable between Pol ε and Pol2core. Singleturnover pre-steady-state kinetics alsoshowed that the kpol of Pol ε and Pol2core werecomparable when pre-loading the polymeraseonto the primer-template before adding Mg2+and dTTP. However, a global fit of the dataover six sequential nucleotide incorporationsrevealed that the overall polymerization rateand processivity was higher for Pol ε than forPol2core. The largest difference was observedwhen challenged for the formation of aternary complex and incorporation of thefirst nucleotide. Pol ε needed less than asecond to incorporate a nucleotide, butseveral seconds passed before Pol2coreincorporated detectable levels of the firstnucleotide. We conclude that the accessorysubunits and the C-terminus of Pol2 do notinfluence the catalytic rate of Pol ε butfacilitate the loading and incorporation of thefirst nucleotide by Pol ε.
Place, publisher, year, edition, pages
USA, 2014. Vol. 290, no 6, 3825-3835 p.
DNA polymerase, DNA repair, DNA replication, enzyme catalysis, enzyme kinetics
Research subject Biochemistry
IdentifiersURN: urn:nbn:se:umu:diva-97692DOI: 10.1074/jbc.M114.615278ISI: 000349456000053PubMedID: 25538242OAI: oai:DiVA.org:umu-97692DiVA: diva2:777575