定位诱变产生的DNA聚合酶β某些特性的改变

Alternations of Some Enzymological Characterizations of Site-mutagenized DNA Polymerase β

作者对定位诱变克隆的大鼠DNA聚合酶β(pol β)基因产物的酶学特性作了初步研究。结果表明Gln代替Arg^(182)(RQ 182)或Arg^(183)(RQ 183)后,变异的酶对单链模板DNA的结合增强。RQ 182对底物dTTP和引物Oligo(dT)的Km值与野生型pol β相似,但RQ 183对底物的Km值较野生型pol β增加了3倍,对引物的Km值也显著增加,表明RQ 183对底物和引物的亲和力明显下降。推测Arg^(182)和Arg^(183)参与pol β的活性中心功能,并与酶和模板DNA结合,对引物的识别以及与底物的结合和催化有关。

The enzymological characterizations of site-mutagenized rat recombinant DNA polymeraseβ, RQ182 and RQ183 were studied The phosphocellulose column chromatographies showed that the mutant and the wild DNA polymerases were all eluted by about 0.5 mol/ L KCI, but the denatured DNA-cellulose chromatographies showed that although the wild enzyme was eluted by 0.35 mol/L KCI, RQ182 and RQ183 were eluted by 0.55 and 0.45 mol/L KCI, respectively, indicating that the binding abilities to DNA of the mutant enzymes were increased. Km values for the substrate (dTTP)of the wild enzyme, RQ182 and RQ183 were determined as 38.5, 34.5 and 111.1 μmol/L, respectively,and the Km values for the primer (oligo(dT)) were 1.28, 1.96 and 6.58 μg/ml, respectively. The results showed that the affinities of RQ183 to the substrate and the primer were decreased dramatically. It is suggested that Arg182 and Arg183 were involved in the active site function of DNA polymerase β, in binding to DNA template, in recognizing of primer, and in binding to and catalyzing of substrates of the enzyme.