Calf thymus DNA polymerase δ Independent of proliferating cell nuclear antigen (PCNA)

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Abstract
DNA polymerase .delta. from calf thymus was purified under conditions that minimized proteolysis to a specific activity of 27,000 units/mg. The four step isolation procedure included phosphocellulose, hydroxyapatite, heparin-Sepharose and FPLC-MonoS. This enzyme consists of four polypeptides with Mr of 140, 125, 48 and 40 kilodaltons. Velocity gradient sedimentation is glycerol removed the 48 kDa polypeptide while the other three sedimented with the DNA polymerase activity. The biochemical properties of the three subunit enzyme and the copurification of 3'' .fwdarw. 5'' exonuclease activity were typical for a bona fide DNA polymerase .delta.. Tryptic peptide analysis showed that the 140 kDa polypeptide was different from the catalytic 180 kDa polypeptide of calf thymus DNA polymerase .alpha.. Both high Mr polypeptides (140 and 125 kDa) were catalytically active as analysed in an activity gel. Four templates were used by DNA polymerase .delta. with different preferences, namely poly(dA)/ oligo(dT)12-18 > > activated DNA > poly(dA-dT) > primed single-stranded M13DNA. Calf thymus proliferating cell nuclear antigen (PCNA) could not stimulated this DNA polymerase .delta. in any step of the isolation procedure. It tested on poly(dA)/oligo(dT)12-18 (base ratio 10:1), PCNA had no stimulatory effect on DNA polymerase .delta. when tested with low enzyme DNA ratio nor did it change the kinetic behaviour of the enzyme. DNA polymerase .delta. itself did not contain PCNA. The enzyme had an intrinsic processivity of several thousand bases, when tested either on the homopolymer poly(dA)/oligo(dT)12-18 (base ratio 64:1) or on primed single-stranded M13DNA. Contrary to DNA polymerase .alpha., no pausing sites were seen with DNA polymerase .delta.. Under optimal in vitro replication conditions the enzyme could convert primed single-stranded circular M13 DNA of 7,200 bases to its double-stranded form in less than 10 min. This supports that a PCNA independent DNA polymerase .delta. exists in calf thymus in addition to a PCNA dependent enzyme (Lee, M.Y.W.T. et al. (1984) Biochemistry 23, 1906-1913).