Mozzherin D J, Fisher P A
Department of Pharmacological Sciences, University Medical Center, State University of New York at Stony Brook 11794-8651, USA.
Biochemistry. 1996 Mar 19;35(11):3572-7. doi: 10.1021/bi952142p.
DNA polymerase epsilon (pol epsilon) was purified to apparent homogeneity from human placentas. The purified enzyme contains a single polypeptide of approximately 170 kDa (apparent mass) and has both DNA polymerase and 3'-5'-exonuclease activities. Competitive inhibition studies indicate that like DNA polymerases alpha and delta (pol alpha and pol delta, respectively), free pol epsilon binds single-stranded but not double-stranded DNA. This conclusion was confirmed by sedimentation binding analysis. Also like pol alpha and pol beta, pol epsilon exhibits induced dNTP inhibition in the presence of template annealed to complementary primer containing a 2',3'-H (dideoxy)-terminus. Together, these data suggest that pol epsilon follows an ordered sequential ter-reactant mechanism of substrate recognition and binding; it binds template first followed by annealed primer and then template-specified dNTP. Enzymologic studies suggest that in contrast to both pol alpha and pol delta, pol epsilon functions more efficiently as gap size decreases. This observation is consistent with a specific role for pol epsilon in gap-filling in vivo. Gap-filling is essential for both replication and repair.
DNA聚合酶ε（pol ε）从人胎盘中纯化至表观均一。纯化后的酶含有一条约170 kDa（表观质量）的单一多肽，具有DNA聚合酶和3'-5'-核酸外切酶活性。竞争性抑制研究表明，与DNA聚合酶α和δ（分别为pol α和pol δ）一样，游离的pol ε结合单链DNA而非双链DNA。沉降结合分析证实了这一结论。同样与pol α和pol β一样，在与含有2',3'-H（双脱氧）末端的互补引物退火的模板存在下，pol ε表现出诱导性dNTP抑制。这些数据共同表明，pol ε遵循底物识别和结合的有序顺序三反应物机制；它首先结合模板，然后是退火引物，接着是模板指定的dNTP。酶学研究表明，与pol α和pol δ不同，随着缺口大小减小，pol ε的功能更有效。这一观察结果与pol ε在体内填补缺口的特定作用一致。填补缺口对于复制和修复都至关重要。
