Sabatino R D, Myers T W, Bambara R A, Kwon-Shin O, Marraccino R L, Frickey P H
Department of Biochemistry, University of Rochester School of Medicine and Dentistry, New York 14642.
Biochemistry. 1988 Apr 19;27(8):2998-3004. doi: 10.1021/bi00408a050.
We have demonstrated that calf thymus DNA polymerases alpha and delta are capable of highly processive DNA synthesis. Processivity values between 300 and 2000 nucleotides were observed when poly(dA)-oligo(dT) or singly primed single-stranded circular bacteriophage M13 DNA at pH 6.0 and 1 mM magnesium chloride was used. These conditions do not correlate with conditions, pH 7.0 and 5 mM magnesium chloride, that support the maximum synthetic rate. Lowering the pH and magnesium concentration lowers the Km value of the reaction with respect to primer terminus concentration. Furthermore, under these same conditions, both polymerases become insensitive to dissociation from the template as a result of encountering the 5' ends of primers. Overall, these results suggest that the affinity of the polymerases for the primer termini is higher throughout the polymerization reaction of pH and magnesium concentrations are lowered from those favoring maximum synthetic rate. Experiments with short primer templates, however, indicate that this higher affinity does not cause the DNA polymerase to remain stably bound after synthesizing up to the end of the template.
我们已经证明，小牛胸腺DNA聚合酶α和δ能够进行高度持续的DNA合成。当使用聚（dA）-寡聚（dT）或单引物单链环状噬菌体M13 DNA，在pH 6.0和1 mM氯化镁条件下时，观察到持续合成值在300至2000个核苷酸之间。这些条件与支持最大合成速率的pH 7.0和5 mM氯化镁条件不相关。降低pH值和镁浓度会降低反应相对于引物末端浓度的Km值。此外，在相同条件下，由于遇到引物的5'末端，两种聚合酶对从模板上解离变得不敏感。总体而言，这些结果表明，在pH值和镁浓度从有利于最大合成速率的值降低的整个聚合反应过程中，聚合酶对引物末端的亲和力更高。然而，使用短引物模板的实验表明，这种更高的亲和力不会导致DNA聚合酶在合成至模板末端后仍稳定结合。
