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

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 in 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'----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 much much greater than activated DNA greater than poly(dA-dT) greater than 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. If 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).

DNA polymerases alpha, delta, and epsilon: three distinct enzymes from HeLa cells.

“…Its catalytic properties are clearly different from those of DNA pol E (or 82), which we have characterized from HeLa cells (8,10) and others have studied from calf thymus (6,7,19,25). The purification of DNA pol and E simultaneously from the same HeLa extract and the differences in the partial peptide maps of their large subunits support the premise that the enzyme proteins are different in spite of the weak cross-reactivity with a polyclonal antibody and possible common motifs in peptide maps found earlier between HeLa DNA pol E and calf thymus DNA pol 8 (11).…”

DNA Ligase I Selectively Affects DNA Synthesis by DNA Polymerases δ and ε Suggesting Differential Functions in DNA Replication and Repair

“…Whereas pol ␦ is processive only in the presence of PCNA, pol ⑀ is very active even in the absence of PCNA. Previous studies have reported that pol ⑀ is completely unresponsive to the addition of PCNA (20,35); however, more detailed kinetic studies have shown that PCNA can also stimulate the processivity of pol ⑀ and that pol ⑀ interacts with PCNA, increasing its rate of nucleotide incorporation (36). In the presence of RF-C and ATP, pol ⑀, like pol ␦, is able to form a stable complex with PCNA at the 3Ј-end of the primer, called pol ⑀ holoenzyme (30,37).…”

Synthesis of DNA containing the simian virus 40 origin of replication by the combined action of DNA polymerases alpha and delta.

“…Recently another polymerase, also called Pol-6, which does not require PCNA, has been isolated from several mammalian sources (17)(18)(19)(20). These different Pol-8 preparations share some common properties, including 3' -+ 5' exonuclease activity and resistance to butylphenyl-dGTP (BuPh-dGTP) and butylanilo-dATP.…”