ABSTRACT
The major role of DNA polymerase ? was thought to be limited in its involvement in short patch base excision repair by removing 5'-deoxyribose phosphate and base insertion. However, the recent researches indicate that polymerase ? might take part in a wide spectrum of DNA metabolism reactions, including long patch base excision repair, DNA replication, recombination, meiosis and transleisional DNA synthesis. Because of its wide and important cellular function, an inappropriate intracellular polymerase ? level might be associated with genomic instability. Down-regulation or mutation of polymerase ? is mutagenic due to deficient in DNA repair, while overexpression of this error-prone ? polymerase might perturb the normal function of other accurate polymerases and cause genomic instability as well.
ABSTRACT
The enzymological characterizations of site-mutagenized rat recombinant DNA polymerase?, 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 ?mol/L, respectively,and the Km values for the primer (oligo(dT)) were 1.28, 1.96 and 6.58 ?g/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 ?, in binding to DNA template, in recognizing of primer, and in binding to and catalyzing of substrates of the enzyme.