Calf thymus DNA polymerase delta independent of proliferating cell nuclear antigen (PCNA).

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).     

PCNA-dependent DNA polymerase delta from rabbit bone marrow.

Proliferating cell nuclear antigen (PCNA) and PCNA-dependent DNA polymerase delta were partially purified and characterized from rabbit bone marrow. Rabbit DNA polymerase delta sediments at 8.2 S upon glycerol density gradient centrifugation. Similar to calf thymus PCNA-dependent DNA polymerase delta, a 125-123-kDa doublet and 48-kDa polypeptides correlate with DNA polymerase activity. Western blotting of rabbit DNA polymerase delta with polyclonal antibody to calf thymus PCNA-dependent DNA polymerase delta gives the same results as calf thymus delta; the 125-123-kDa doublet is recognized. PCNA-dependent DNA polymerase delta is resistant to inhibition by dideoxynucleotides and is relatively insensitive to inhibition by N2-[p-(n-butyl)phenyl]dGTP. A 3'-->5' exonuclease copurifies with the DNA polymerase. The processivity of DNA polymerase delta alone is very low but greatly increases with the addition of PCNA from rabbit bone marrow or calf thymus. Comparative studies of the original DNA polymerase delta from rabbit bone marrow demonstrate a lack of recognition by antibodies to calf thymus delta and a high degree of processivity in the absence of PCNA. Additionally, the originally described DNA polymerase delta is a single polypeptide of 122 kDa. These features would recategorize the original delta to the epsilon category by recently proposed convention. PCNA-dependent DNA polymerase delta is a relatively minor component of rabbit bone marrow compared to DNA polymerase alpha and PCNA-independent DNA polymerase delta (epsilon), the relative proportions being alpha, 60%; delta, 7%; and epsilon, 30%.     

Further studies on calf thymus DNA polymerase delta purified to homogeneity by a new procedure

DNA polymerase delta from calf thymus has been purified to apparent homogeneity by a new procedure which utilizes hydrophobic interaction chromatography with phenyl-Sepharose at an early step to separate most of the calcium-dependent protease activity from DNA polymerase delta and alpha. The purified enzyme migrates as a single protein band on polyacrylamide gel electrophoresis under nondenaturing conditions. The sedimentation coefficient of the enzyme is 7.9 S, and the Stokes radius is 53 A. A molecular weight of 173K has been calculated for the native enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the homogeneous enzyme reveals two polypeptides of 125 and 48 kDa. This subunit structure differs from that of DNA polymerase delta prepared by our previous procedure, which was composed of subunits of 60 and 49 kDa [Lee, M. Y. W. T., Tan, C.-K., Downey , K. M., & So, A. G. (1981) Prog . Nucleic Acid Res. Mol. Biol. 26, 83-96], suggesting that the 60-kDa polypeptide may have been derived from the 125-kDa polypeptide during enzyme purification, possibly as the result of cleavage of an unusually sensitive peptide bond. DNA polymerase delta is separated from DNA polymerase alpha by hydrophobic interaction chromatography on phenyl-Sepharose; DNA polymerase delta is eluted at pH 7.2 and DNA polymerase alpha at pH 8.5. DNA polymerase delta can also be separated from DNA polymerase alpha by chromatography on hydroxylapatite; DNA polymerase alpha binds to hydroxylapatite in the presence of 0.5 M KCl, whereas DNA polymerase delta is eluted at 90 mM KCl.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of human DNA polymerase delta and its immunochemical relationships with DNA polymerase alpha and epsilon.

DNA polymerase delta was purified from human placenta and its polymerase catalytic subunit identified as a 125-kDa polypeptide by activity staining. This 125-kDa form of DNA polymerase delta resembles that reported from calf thymus (Lee, M. Y. W. T., Tan, C.-K., Downey, K. M., and So, A. G. (1984) Biochemistry 23, 1906-1913) and differs in molecular properties from a previously described form isolated from human placenta (Lee, M. Y. W. T., and Toomey, N. L. (1987) Biochemistry 26, 1076-1085) and now referred to as DNA polymerase epsilon. The properties of DNA polymerase delta were further investigated to determine its relationships with DNA polymerase epsilon. The two enzymes differed in their response to proliferating cell nuclear antigen. Monoclonal antibodies against DNA polymerase delta were raised and used to examine its immunochemical relationships with DNA polymerase alpha and epsilon. These studies provided evidence that all three proteins are structurally distinct but share a common epitope(s). Immunofluorescence microscopy indicates that DNA polymerase delta and possibly also DNA polymerase epsilon are localized to the nucleus.     

The human homologue of fission Yeast cdc27, p66, is a component of active human DNA polymerase delta

An essential eukaryotic DNA polymerase, DNA polymerase delta (pol delta), synthesizes DNA processively in the presence of proliferating cell nuclear antigen (PCNA). Recently, a 66 kDa polypeptide (p66) that displays significant homology within its PCNA binding domain to that of fission yeast cdc27 was identified as a component of mouse and calf thymus pol delta. Our studies show that p66 interacts tightly with other subunits of pol delta during size fractionation of human cell extracts, and co-immunoprecipitates with these subunits along with PCNA-dependent polymerase activity. Active human pol delta could be reconstituted by co-expressing p125, p50, and p66 recombinant baculoviruses, but not by co-expressing p125 and p50 alone. Interaction studies demonstrated that p66 stabilizes the association between p125 and p50. Pull-down assays with PCNA-linked beads demonstrated that p66 increases the overall affinity of pol delta for PCNA. These results indicate that p66 is a functionally important subunit of human pol delta that stabilizes the pol delta complex and increases the affinity of pol delta for PCNA.     

Identification and tryptic cleavage of the catalytic core of HeLa and calf thymus DNA polymerase epsilon

DNA polymerase epsilon, formerly known as a proliferating cell nuclear antigen-independent form of DNA polymerase delta, has been shown elsewhere to be catalytically and structurally distinct from DNA polymerase delta. The catalytic activity of HeLa DNA polymerase epsilon, an enzyme consisting of greater than 200- and 55-kDa polypeptides, was assigned to the larger polypeptide by polymerase trap reaction. This catalytic polypeptide was cleaved by incubation with trypsin into two polypeptide fragments with molecular masses of 122 and 136 kDa, the former of which was relatively resistant to further proteolysis and possessed the polymerase activity. The cleavage increased the polymerase and exonuclease activities of the enzyme some 2-3-fold. DNA polymerase epsilon was also purified in a smaller 140-kDa form from calf thymus. The digestion of this form of the enzyme by trypsin also generated a 122-kDa polypeptide. These results suggest that the catalytic core of DNA polymerase epsilon is a 258-kDa polypeptide that is composed of two segments linked with a protease-sensitive area. One of the segments harbors both DNA polymerase and 3'----5' exonuclease activities. In spite of the different polypeptide structures, the catalytic properties of the HeLa enzyme, its trypsin-digested form, and the calf thymus enzyme remained essentially the same.     

Direct interaction of proliferating cell nuclear antigen with the small subunit of DNA polymerase delta

The interaction between proliferating cell nuclear antigen (PCNA) and DNA polymerase delta is essential for processive DNA synthesis during DNA replication/repair; however, the identity of the subunit of DNA polymerase delta that directly interacts with PCNA has not been resolved until now. In the present study we have used reciprocal co-immunoprecipitation experiments to determine which of the two subunits of core DNA polymerase delta, the 125-kDa catalytic subunit or the 50-kDa small subunit, directly interacts with PCNA. We found that PCNA co-immunoprecipitated with human p50, as well as calf thymus DNA polymerase delta heterodimer, but not with p125 alone, suggesting that PCNA directly interacts with p50 but not with p125. A PCNA-binding motif, similar to the sliding clamp-binding motif of bacteriophage RB69 DNA polymerase, was identified in the N terminus of p50. A 22-amino acid oligopeptide containing this sequence (MRPFL) was shown to bind PCNA by far Western analysis and to compete with p50 for binding to PCNA in co-immunoprecipitation experiments. The binding of p50 to PCNA was inhibited by p21, suggesting that the two proteins compete for the same binding site on PCNA. These results establish that the interaction of PCNA with DNA polymerase delta is mediated through the small subunit of the enzyme.     

The small subunit is required for functional interaction of DNA polymerase delta with the proliferating cell nuclear antigen.

DNA polymerase delta is usually isolated as a heterodimer composed of a 125 kDa catalytic subunit and a 50 kDa small subunit of unknown function. The enzyme is distributive by itself and requires an accessory protein, the proliferating cell nuclear antigen (PCNA), for highly processive DNA synthesis. We have recently demonstrated that the catalytic subunit of human DNA polymerase delta (p125) expressed in baculovirus-infected insect cells, in contrast to the native heterodimeric calf thymus DNA polymerase delta, is not responsive to stimulation by PCNA. To determine whether the lack of response to PCNA of the recombinant catalytic subunit is due to the absence of the small subunit or to differences in post-translational modification in insect cells versus mammalian cells, we have co-expressed the two subunits of human DNA polymerase delta in insect cells. We have demonstrated that co-expression of the catalytic and small subunits of human DNA polymerase delta results in formation of a stable, fully functional heterodimer, that the recombinant heterodimer, similar to native heterodimer, is markedly stimulated (40- to 50-fold) by PCNA and that the increase in activity seen in the presence of PCNA is the result of an increase in processivity. These data establish that the 50 kDa subunit is essential for functional interaction of DNA polymerase delta with PCNA and for highly processive DNA synthesis.     

Identification of a fourth subunit of mammalian DNA polymerase delta

A 12-kDa and two 25-kDa polypeptides were isolated with highly purified calf thymus DNA polymerase delta by conventional chromatography. A 16-mer peptide sequence was obtained from the 12-kDa polypeptide which matched a new open reading frame from a human EST () encoding a hypothetical protein of unknown function. The protein was designated as p12. Human EST was identified as the putative human homologue of Schizosaccharomyces pombe Cdm1 by a tBlastn search of the EST data base using S. pombe Cdm1. The open reading frame of human EST encoded a polypeptide of 107 amino acids with a predicted molecular mass of 12.4 kDa, consistent with the experimental findings. p12 is 25% identical to S pombe Cdm1. Both of the 25-kDa polypeptide sequences matched the hypothetical KIAA0039 protein sequence, recently identified as the third subunit of pol delta. Western blotting of immunoaffinity purified calf thymus pol delta revealed the presence of p125, p50, p68 (the KIAA0039 product), and p12. With the identification of p12 mammalian pol delta can now be shown to consist of four subunits. These studies pave the way for more detailed analysis of the possible functions of the mammalian subunits of pol delta.     

Mammalian DNA ligases. Biosynthesis and intracellular localization of DNA ligase I

Mammalian DNA ligase I is presumed to act in DNA replication. Rabbit antibodies against the homogeneous enzyme from calf thymus inhibited DNA ligase I activity and consistently recognized a single polypeptide of 125 kDa when cells from an established bovine kidney cell line (MDBK) were lysed rapidly by a variety of procedures and subjected to immunoblotting analysis. After biosynthetic labeling of MDBK cells with [35S]methionine, immunoprecipitation experiments revealed a polypeptide of 125 kDa that did not appear when purified calf thymus DNA ligase I was used in competition. A 125-kDa polypeptide was adenylated when immunoprecipitated protein from MDBK cells was incubated with [alpha-32P]ATP. Thus, the apparent molecular mass of the initial translation product is identical or nearly so to that of the purified enzyme. The half-life of the protein is 7 h as determined by pulse-chase experiments in asynchronous MDBK cells. Immunocytochemistry and indirect immunofluorescence experiments showed that DNA ligase I is localized to cell nuclei.     

Purification and characterization of DNA polymerase II from the yeast Saccharomyces cerevisiae. Identification of the catalytic core and a possible holoenzyme form of the enzyme

We have purified yeast DNA polymerase II to near homogeneity as a 145-kDa polypeptide. During the course of this purification we have detected and purified a novel form of DNA polymerase II that we designate as DNA polymerase II. The most highly purified preparations of DNA polymerase II are composed of polypeptides with molecular masses of 200, 80, 34, 30, and 29 kDa. Immunological analysis and peptide mapping of DNA polymerase II and the 200-kDa subunit of DNA polymerase II indicate that the 145-kDa DNA polymerase II polypeptide is derived from the 200-kDa polypeptide of DNA polymerase II. Activity gel analysis shows that the 145- and the 200-kDa polypeptides have catalytic function. The polypeptides present in the DNA polymerase II preparation copurify with the polymerase activity with a constant relative stoichiometry during chromatography over five columns and co-sediment with the activity during glycerol gradient centrifugation, suggesting that this complex may be a holoenzyme form of DNA polymerase II. Both forms of DNA polymerase II possess a 3'-5' exonuclease activity that remains tightly associated with the polymerase activity during purification. DNA polymerase II is similar to the proliferating cell nuclear antigen (PCNA)-independent form of mammalian DNA polymerase delta in its resistance to butylpheny-dGTP, template specificity, stimulation of polymerase and exonuclease activity by KCl, and high processivity. Although calf thymus PCNA does not stimulate the activity of DNA polymerase II on poly(dA):oligo(dT), possibly due to the limited length of the template, the high processivity of yeast DNA polymerase II on this template can be further increased by the addition of PCNA, suggesting that conditions may exist for interactions between PCNA and yeast DNA polymerase II.     

The PCNA from Thermococcus fumicolans functionally interacts with DNA polymerase delta

We have cloned the gene encoding proliferating cell nuclear antigen (PCNA) from the hyperthermophilic euryarchaeote Thermococcus fumicolans (Tfu). Tfu PCNA contains 250 amino acids with a calculated M(r) of 28,000 and is 26% identical to human PCNA. Next, Tfu PCNA was overexpressed in Escherichia coli and it showed an apparent molecular mass of 33.5 kDa. The purified Tfu PCNA was tested first with recombinant Tfu DNA polymerase I (Tfu pol) and second with calf thymus DNA polymerase delta (pol delta). When tested with the homologous Tfu pol on bacteriophage lambda DNA, large amounts of Tfu PCNA were required to obtain two- to threefold stimulation. Surprisingly, however, Tfu PCNA was much more efficient than human PCNA in stimulating calf thymus pol delta. Our data suggest that PCNA has been functionally conserved not only within eukaryotes but also from hyperthermophilic euryarchaeotes to mammals.     

Evidence that DNA polymerase delta isolated by immunoaffinity chromatography exhibits high-molecular weight characteristics and is associated with the KIAA0039 protein and RPA

DNA polymerase delta, the key enzyme for eukaryotic chromosomal replication, has been well characterized as consisting of a core enzyme of a 125 kDa catalytic subunit and a smaller 50 kDa subunit. However, less is known about the other proteins that may comprise additional subunits or participate in the macromolecular protein complex that is involved in chromosomal DNA replication. In this study, the properties of calf thymus pol delta preparations isolated by immunoaffinity chromatography were investigated. It is demonstrated for the first time using highly purified preparations that the pol delta heterodimer is associated with other polypeptides in high-molecular weight species that range from 260000 to >500000 in size, as determined by FPLC gel filtration. These preparations are associated with polypeptides of ca. 68-70, 34, 32, and 25 kDa. Similar findings were revealed with glycerol gradient ultracentrifugation. The p68 polypeptide was shown to be a PCNA binding protein by overlay methods with biotinylated PCNA. Protein sequencing of the p68, p34, and p25 polypeptide bands revealed sequences that correspond to the hypothetical protein KIAA0039. KIAA0039 displays a small but significant degree of homology to Schizosaccharomyces pombe Cdc27, which, like Saccharomyces cerevisiae Pol32p, has been described as the third subunit of yeast pol delta. These studies provide evidence that p68 is a subunit of pol delta. In addition, the p68-70 and p32 polypeptides were found to be derived from the 70 and 32 kDa subunits of RPA, respectively.     

Immunochemical analysis of molecular forms of mammalian DNA ligases I and II

Using specific antibodies against calf thymus DNA ligases I and II (EC 6.5.1.1), we have investigated the polypeptide structures of DNA ligases I and II present in the impure enzyme preparations, and estimated the polypeptides of DNA ligases I and II present in vivo. Immunoblot analysis of DNA ligase I after sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a 130-kDa polypeptide as a major one in the enzyme preparations from calf thymus throughout the purification. In addition to the 130-kDa polypeptide, a 200-kDa polypeptide was detected in the enzyme preparations at the earlier steps of the purification, and a 90-kDa polypeptide was observed as a minor one in the enzyme preparations at the later steps of the purification. The polypeptides with molecular weight of 130 000 and 90 000 were detected by SDS-polyacrylamide gel electrophoresis of DNA ligase I-[3H]AMP complex. These results suggest that a 200-kDa polypeptide of DNA ligase I present in vivo is degraded to a 130-kDa polypeptide and then to a 90-kDa polypeptide during the isolation and purification procedures. On the other hand, the monospecific antibody against calf thymus DNA ligase II cross-reacted with only a 68 kDa polypeptide in the enzyme preparations throughout the purification, suggesting that the 68-kDa polypeptide is a single form of calf thymus DNA ligase II present in vivo as well as in vitro.     

Mammalian DNA ligases. Catalytic domain and size of DNA ligase I.

DNA ligase I is the major DNA ligase activity in proliferating mammalian cells. The protein has been purified to apparent homogeneity from calf thymus. It has a monomeric structure and a blocked N-terminal residue. DNA ligase I is a 125-kDa polypeptide as estimated by sodium dodecyl sulfate-gel electrophoresis and by gel chromatography under denaturing conditions, whereas hydrodynamic measurements indicate that the enzyme is an asymmetric 98-kDa protein. Immunoblotting with rabbit polyclonal antibodies to the enzyme revealed a single polypeptide of 125 kDa in freshly prepared crude cell extracts of calf thymus. Limited digestion of the purified DNA ligase I with several reagent proteolytic enzymes generated a relatively protease-resistant 85-kDa fragment. This domain retained full catalytic activity. Similar results were obtained with partially purified human DNA ligase I. The active large fragment represents the C-terminal part of the intact protein, and contains an epitope conserved between mammalian DNA ligase I and yeast and vaccinia virus DNA ligases. The function of the N-terminal region of DNA ligase I is unknown.     

Purification and characterization of delta helicase from fetal calf thymus.

A DNA helicase (delta helicase) which partially copurifies with DNA polymerase delta has been highly purified from fetal calf thymus. delta helicase differs in physical and enzymatic properties from other eukaryotic DNA helicases described thus far. The enzyme has an apparent mass of 57 kDa by gel filtration and is associated with polypeptides of 56 and 52 kDa by SDS-polyacrylamide gel electrophoresis. Photo-cross-linking of the purified enzyme with [alpha-32P]ATP resulted in labeling of a polypeptide of approximately 58 kDa, suggesting that the active site is present on the larger polypeptide. Unwinding of a partial duplex requires a nucleoside triphosphate which can be either ATP or dATP but not a nonhydrolyzable analogue of ATP. Other ribo- and deoxyribonucleoside triphosphates have little or no activity as cofactors. delta helicase also has DNA-dependent ATPase activity which has a relatively low Km for ATP (40 microM). delta helicase binds to single-stranded DNA but has little or no affinity for double-stranded DNA or single-stranded RNA. Similar to replicative DNA helicases from prokaryotes and the herpes simplex virus type 1 helicase-primase, delta helicase translocates in the 5'-3' direction along the strand to which it is bound and preferentially unwinds DNA substrates with a forklike structure.     

Reconstitution and characterization of the human DNA polymerase delta four-subunit holoenzyme

Mammalian DNA polymerase delta was originally characterized as a tightly associated heterodimer consisting of the catalytic subunit, p125, and the p50 subunit. Recently, two additional subunits, the third (p68) and fourth subunits (p12), have been identified. The heterotetrameric human pol delta complex was reconstituted by overexpression of the four subunits in Sf9 cells, followed by purification to near-homogeneity using FPLC chromatography. The properties of the four-subunit enzyme were shown to be functionally indistinguishable from those of pol delta isolated from calf thymus. The physicochemical properties of both the reconstituted heterotetramer and the heterodimer of the p125 and p50 subunits were examined by gel filtration and glycerol gradient ultracentrifugation. These studies show quite clearly that the heterodimer and heterotetramer complexes do not behave in solution as dimeric structures. This issue is of significance because several studies of the yeast pol delta complexes have indicated that the third subunit is able to bring about the dimerization of the pol delta complex. The heterodimer is only weakly stimulated by PCNA, whereas the heterotetramer is strongly stimulated to a level with a specific activity comparable to that of the calf thymus enzyme. These results resolve earlier, conflicting reports on the response of the heterodimer to PCNA. Nevertheless, the heterodimer does have some ability to interact functionally with PCNA, consistent with evidence that the p125 subunit itself has an ability to interact with PCNA. The functional interaction of PCNA with the pol delta complex may likely involve multiple contacts.     

Immunochemical detection of a primase activity related subunit of DNA polymerase alpha from human and mouse cells using the monoclonal antibody

A hybrid cell line (HDR-854-E4) secreting monoclonal antibody (E4 antibody) against a subunit of human DNA polymerase alpha was established by immunizing mice with DNA replicase complex (DNA polymerase alpha-primase complex) prepared from HeLa cells. The E4 antibody immunoprecipitates DNA replicase complex from both human and mouse cells. The E4 antibody neutralizes the primase activity as assessed either by the direct primase assay (incorporation of [alpha-32P]AMP) or by assay of DNA polymerase activity coupled with the primase activity using unprimed poly(dT) as a template. The E4 antibody does not neutralize DNA polymerase alpha activity with the activated calf thymus DNA as a template. Western immunoblotting analysis shows that the E4 antibody binds to a polypeptide of 77 kilodaltons (kDa) which is tightly associated with DNA polymerase alpha. The 77-kDa polypeptide was distinguished from the catalytic subunit (160 and 180 kDa) for DNA synthesis which was detected by another monoclonal antibody, HDR-863-A5. Furthermore, it is unlikely that the 77-kDa peptide is the primase, since we found that the E4 antibody also immunoprecipitates the mouse 7.3S DNA polymerase alpha which has no primase activity, and Western immunoblotting analysis shows that the 77-kDa polypeptide is a subunit of the 7.3S DNA polymerase alpha. Furthermore, after dissociation of the primase from mouse DNA replicase by chromatography on a hydroxyapatite column in the presence of dimethyl sulfoxide and ethylene glycol, the 77-kDa polypeptide is associated with DNA polymerase alpha, and not with the primase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calf thymus RF-C as an essential component for DNA polymerase delta and epsilon holoenzymes function.

By using a complementation assay that enabled DNA polymerase delta and DNA polymerase epsilon to replicate a singly-DNA primed M13 DNA in the presence of proliferating cell nuclear antigen (PCNA) and Escherichia coli single-stranded DNA binding protein (SSB), we have purified from calf thymus in a five step procedure a multipolypeptide complex with molecular masses of polypeptides of 155, 70, 60, 58, 39 (doublet), 38 (doublet) and 36 kDa. The protein is very likely replication factor C (Tsurimoto, T. and Stillman, B. (1989) Mol. Cell. Biol. 9, 609-619). This conclusion is based on biochemical and physicochemical data and the finding that it contains a DNA stimulated ATPase which is under certain conditions stimulated by PCNA. Together RF-C, PCNA and ATP convert DNA polymerases delta and epsilon to holoenzyme forms, which were able to replicate efficiently SSB-covered singly-DNA primed M13 DNA. Calf thymus RF-C could form a primer recognition complex on a 3'-OH primer terminus in the presence of calf thymus PCNA and ATP. Holoenzyme complexes of DNA polymerase delta and epsilon could be isolated suggesting that these enzymes directly interact with the auxiliary proteins in a similar way. Under optimal replication conditions on singly-DNA primed M13 DNA the DNA synthesis rate of DNA polymerase delta was higher than of DNA polymerase epsilon. Based on these functional date possible roles of these two DNA polymerases in eukaryotic DNA replication are discussed.     

Isolation of the DNA polymerase alpha core enzyme from mouse cells

DNA polymerase alpha has been purified from mouse hybridoma cells approximately 30,000-fold using a combination of conventional and high performance liquid chromatography. In contrast to previous characterizations of mammalian DNA polymerase alpha, this enzyme has a single high molecular mass polypeptide (185 kDa) in tight association with a 68-kDa polypeptide and this structure appears to be the core DNA polymerase of the mouse cells. The biochemically purified enzyme, with a specific activity of approximately 200,000 units/mg protein, has an estimated molecular mass by gel filtration chromatography of 240 kDa and sedimentation value of 9 S, consistent with the enzyme being a heterodimer of 185 and 68 kDa. The enzyme is sensitive to both N-ethylmaleimide and aphidicolin and insensitive to ddTTP. Using an activated DNA template, the apparent Km values for the deoxynucleotide triphosphates are approximately 0.5-1 microM. The purified DNA polymerase has neither exonuclease nor primase activities and is the predominant DNA polymerase alpha activity in the mouse cells.