Primary structure of the catalytic subunit of calf thymus DNA polymerase delta: sequence similarities with other DNA polymerases.

The 125- and 48-kDa subunits of bovine DNA polymerase delta have been isolated by SDS-polyacrylamide gel electrophoresis and demonstrated to be unrelated by partial peptide mapping with N-chlorosuccinimide. A 116-kDa polypeptide, usually present in DNA polymerase delta preparations, was shown to be a degraded form of the 125-kDa catalytic subunit. Amino acid sequence data from Staphylococcus aureus V8 protease, cyanogen bromide, and trypsin digestion of the 125- and 116-kDa polypeptides were used to design primers for the polymerase chain reaction to determine the nucleotide sequence of a full-length cDNA encoding the catalytic subunit of bovine DNA polymerase delta. The predicted polypeptide is 1106 amino acids in length with a calculated molecular weight of 123,707. This is in agreement with the molecular weight of 125,000 estimated from SDS-polyacrylamide gel electrophoresis. Comparison of the deduced amino acid sequence of the catalytic subunit of bovine DNA polymerase delta with that of its counterpart from Saccharomyces cerevisiae showed that the proteins are 44% identical. The catalytic subunit of bovine DNA polymerase delta contains the seven conserved regions found in a number of bacterial, viral, and eukaryotic DNA polymerases. It also contains five additional regions that are highly conserved between bovine and yeast DNA polymerase delta, but these regions share little or no homology with the alpha polymerases. Four of these additional regions are also highly homologous to the herpes virus family of DNA polymerases, whereas one region is not homologous to any other DNA polymerase that has been sequenced thus far.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human placental DNA polymerase delta: identification of a 170-kilodalton polypeptide by activity staining and immunoblotting

DNA polymerase delta was isolated from human placenta and identified as such on the basis of its association with a 3'- to 5'-exonuclease activity. The association of the polymerase and exonuclease activities was maintained throughout purification and attempted separations by physical or electrophoretic methods. Moreover, ratios of the two activities remained constant during the purification steps, and both activities were inhibited by aphidicolin, oxidized glutathione, and N-ethylmaleimide. The purified enzyme had an estimated molecular weight of 172,000, on the basis of a Stokes radius of 53.6 A and a sedimentation coefficient of 7.8 S. On sodium dodecyl sulfate (SDS) gel electrophoresis, polymerase delta preparations contained a band of ca. 170 kilodaltons (kDa) as well as several smaller polypeptides. The 170-kDa polypeptide was identified as the largest polypeptide component in the preparation possessing DNA polymerase activity by an activity staining procedure following gel electrophoresis in the presence of SDS. Western blotting of DNA polymerase delta with polyclonal antisera also revealed a single 170-kDa immunoreactive polypeptide. Monoclonal antibodies to KB cell polymerase alpha inhibited placental polymerase alpha but did not inhibit DNA polymerase delta, while the murine polyclonal antisera to polymerase delta inhibited delta but not alpha. These findings establish the existence of DNA polymerase delta in a human tissue and support the view that both its polymerase and its exonuclease activities may be associated with a single protein.     

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.     

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.     

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%.     

Isolation of multiple forms of DNA polymerase delta: evidence of proteolytic modification during isolation

The subunit structures of a number of human placenta DNA polymerase delta preparations were investigated by Western blotting with polyclonal antisera and by activity staining following polyacrylamide gel electrophoresis. When immunoblots and activity stains were performed on different enzyme preparations, putative catalytic subunits of (a) 170, (b) 120, or (c) 50-70 kilodaltons (kDa) were observed. It was also observed that the lower molecular weight forms could be generated upon storage of the preparations. Western blotting of human placental tissue extracts showed that the major immunoreactive polypeptide was 160-170 kDa. Treatment of the extracts with trypsin or Staphylococcus aureus V8 protease led to the generation of immunoreactive polypeptides of lower molecular weights. These studies suggest that the 120-kDa and lower forms of the enzyme are generated via uncontrolled proteolysis and provide a rationale for the observation of different apparent subunit structures previously reported for DNA polymerase delta. In addition, these findings suggest that DNA polymerase delta has a catalytic domain which resides in a protease-resistant domain.     

DNA repair synthesis in human fibroblasts requires DNA polymerase delta

When UV-irradiated cultured diploid human fibroblasts were permeabilized with Brij-58 then separated from soluble material by centrifugation, conservative DNA repair synthesis could be restored by a soluble factor obtained from the supernatant of similarly treated HeLa cells. Extensive purification of this factor yielded a 10.2 S, 220,000-dalton polypeptide with the DNA polymerase and 3'- to 5'-exonuclease activities reported for DNA polymerase delta II (Crute, J. J., Wahl, A. F., and Bambara, R. A. (1986) Biochemistry 25, 26-36). Monoclonal antibody to KB cell DNA polymerase alpha, while binding to HeLa DNA polymerase alpha, did not bind to the HeLa DNA polymerase delta. Moreover, at micromolar concentrations N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP) and 2-(p-n-butylanilino)-2'-deoxyadenosine 5'-triphosphate (BuAdATP) were potent inhibitors of DNA polymerase alpha, but did not inhibit the DNA polymerase delta. Neither purified DNA polymerase alpha nor beta could promote repair DNA synthesis in the permeabilized cells. Furthermore, under conditions which inhibited purified DNA polymerase alpha by greater than 90%, neither monoclonal antibodies to DNA polymerase alpha, BuPdGTP, nor BuAdATP was able to inhibit significantly the DNA repair synthesis mediated by the DNA polymerase delta. Thus, it appears that a major portion of DNA repair synthesis induced by UV irradiation might be catalyzed by DNA polymerase delta. When xeroderma pigmentosum human diploid fibroblasts were utilized, DNA repair synthesis dependent upon ultraviolet light could be restored by addition of both T4 endonuclease V and DNA polymerase delta, but not by addition of either one alone. This result suggests that cytosol-depleted permeabilized DNA repair-defective human fibroblasts and HeLa DNA polymerase delta might be exploited to provide a functional assay for purifying active DNA repair factors from DNA repair-proficient cells without a preknowledge of their function.     

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.     

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)     

DNA polymerases alpha and delta are immunologically and structurally distinct

The relationship between DNA polymerases alpha and delta are evaluated immunologically by monoclonal antibody specifically against DNA polymerase alpha and murine polyclonal antiserum against calf thymus DNA polymerase delta. DNA polymerases alpha and delta are found to be immunologically distinct. The structural relationship between the proliferating cell nuclear antigen (PCNA)-dependent calf DNA polymerase delta and DNA polymerase alpha from human and calf was analyzed by two-dimensional tryptic peptide mapping of the catalytic polypeptides. The results demonstrate that the catalytic polypeptides of the PCNA-dependent calf polymerase delta and DNA polymerase alpha are distinct, unrelated, and do not share any common structural determinants. The immunological and structural relationship between a recently identified PCNA-independent form of DNA polymerase delta from HeLa cells was also assessed. This PCNA-independent human polymerase delta was found to be immunologically unrelated to human polymerase alpha but to share some immunological and structural determinants with the PCNA-dependent calf thymus polymerase delta.     

Two forms of DNA polymerase delta from mouse cells. Purification and properties

A procedure is described for the purification from cultured mouse cells of two DNA polymerase "delta-like" enzymes, as defined by intrinsic 3'-exonuclease activity, inhibition by aphidicolin, and relative insensitivity to N2-(p-n-butylphenyl)-dGTP. One of the two enzymes has been purified to near homogeneity and, similar to the DNA polymerase delta from calf thymus described by Lee et al. (Lee, M. Y. W. T., Tan, C. K., Downey, K. M., and So, A. G. (1984) Biochemistry 23, 1906-1913), it has a total molecular mass of 178 kDa (from sedimentation velocity of 8.0 S and Stokes radius of 54 A) and is composed of one each of 125- and 50-kDa polypeptides. It also resembles the DNA polymerase delta of Lee et al. in being stimulated by proliferating cell nuclear antigen (PCNA). It is the first clear structural and functional counterpart of the calf thymus enzyme. The major difference between the mouse DNA polymerase delta and the calf thymus enzyme of Lee et al. is that, under specific conditions, the mouse enzyme is active with poly(dA).oligo(dT) in the absence of PCNA, whereas the activity of the calf thymus enzyme with this template is reported to be completely dependent on PCNA. The reason for this difference is not known at this time. The second mouse cell enzyme has a molecular mass of 112 kDa (from sedimentation velocity of 6.3 S and Stokes radius of 43.0 A) and consists of a single polypeptide of 123-125 kDa in denaturing gels (p125). On the basis of its apparent formation by dissociation of DNA polymerase delta, and multiple similarities with DNA polymerase delta in enzymatic properties, the p125 is provisionally identified as the 125-kDa polypeptide of DNA polymerase delta. The p125 does not respond to PCNA, suggesting that the 50-kDa polypeptide is required for the stimulation of DNA polymerase delta by PCNA. The presence of the p125 in cell extracts would explain reports that DNA polymerase delta consists of a single polypeptide of approximately 125 kDa and/or thast it has a smaller molecular mass than DNA polymerase delta of Lee et al. and is not affected by PCNA (this does not apply to PCNA-independent DNA polymerase delta-like enzymes with higher molecular mass than the polymerase delta of Lee et al., which have recently been named DNA polymerases epsilon).     

Purification and characterization of the 180- and 86-kilodalton subunits of the Saccharomyces cerevisiae DNA primase-DNA polymerase protein complex. The 180-kilodalton subunit has both DNA polymerase and 3'----5'-exonuclease activities.

The yeast Saccharomyces cerevisiae catalytic DNA polymerase I 180-kDa subunit and the tightly associated 86-kDa polypeptide have been purified using immunoaffinity chromatography, permitting further characterization of the DNA polymerase activity of the DNA primase-DNA polymerase protein complex. The subunits were purified to apparent homogeneity from separate overproducing yeast strains using monoclonal antibodies specifically recognizing each subunit. When the individual subunits were recombined in vitro a p86p180 physical complex formed spontaneously, as judged by immunoprecipitation of 180-kDa polypeptide and DNA polymerase activity with the anti-86-kDa monoclonal antibody. The 86-kDa subunit stabilized the DNA polymerase activity of the 180-kDa catalytic subunit at 30 degrees C, the physiological temperature. The apparent DNA polymerase processivity of 50-60 nucleotides on poly(dA).oligo(dT)12 or poly(dT).oligo(A)8-12 template-primer was not affected by the presence of the 86-kDa subunit but was reduced by increased Mg2+ concentration. The Km of the catalytic 180-kDa subunit for dATP or DNA primer terminus was unaffected by the presence of the 86-kDa subunit. The isolated 180-kDa polypeptide was sufficient to catalyze all the DNA synthesis that had been observed previously in the DNA primase-DNA polymerase protein complex. The 180-kDa subunit possessed a 3'----5'-exonuclease activity that catalyzed degradation of polynucleotides, but degradation of oligonucleotide substrates of chain lengths up to 50 was not detected. This exonuclease activity was unaffected by the presence of the 86-kDa subunit. Despite the striking physical similarity of the DNA primase-DNA polymerase protein complex in all eukaryotes examined, the data presented here indicate differences in the enzymatic properties detected in preparations of the DNA polymerase subunits isolated from S. cerevisiae as compared with the properties of preparations from Drosophila cells. In particular, the 3'----5'-exonuclease activity associated with the yeast catalytic DNA polymerase subunit was not masked by the 86-kDa subunit.     

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).     

Identification of DNA polymerase delta in CV-1 cells: studies implicating both DNA polymerase delta and DNA polymerase alpha in DNA replication

DNA polymerases delta and alpha were purified from CV-1 cells, and their sensitivities to the inhibitors aphidicolin, (p-n-butylphenyl)deoxyguanosine triphosphate (BuPdGTP), and monoclonal antibodies directed against DNA polymerase alpha were determined. The effects of these inhibitors on DNA replication in permeabilized CV-1 cells were studied to investigate the potential roles of polymerases delta and alpha in DNA replication. Aphidicolin was shown to be a more potent inhibitor of DNA replication than of DNA polymerase alpha or delta activity. Inhibition of DNA replication by various concentrations of BuPdGTP was intermediate between inhibition of purified polymerase alpha or delta activity. Concentrations of BuPdGTP which totally abolished DNA polymerase alpha activity were much less effective in reducing DNA replication, as well as the activity of DNA polymerase delta. Monoclonal antibodies which specifically inhibited polymerase alpha activity reduced, but did not abolish, DNA replication in permeable cells. BuPdGTP, as well as anti-polymerase alpha antibodies, inhibited DNA replication in a nonlinear manner as a function of time. Depending upon the initial or final rates of inhibition of replication by BuPdGTP and anti-alpha antibodies, as little as 50%, or as much as 80%, of the replication activity can be attributed to polymerase alpha. The remaining replication activity (20-50%) is tentatively attributed to polymerase delta, because it was aphidicolin sensitive and resistant to both anti-polymerase alpha antibodies and low concentrations of BuPdGTP. A concentration of BuPdGTP which abolished polymerase alpha activity reduced, but did not abolish, both the synthesis and maturation of nascent DNA fragments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunological analysis of the polypeptide structure of calf thymus DNA polymerase-primase complex

Five major polypeptides are found in immunoaffinity-purified calf thymus DNA polymerase-DNA primase complex: 185, 160, 68, 55, and 48 kDa. Individual polypeptides purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were used to produce antibodies in rabbits to aid in identifying the relationships between these polypeptides by immunoblotting and enzyme neutralization procedures. Immunoblot analyses showed that the 160-kDa peptide is derived from the 185-kDa peptide and the 48-kDa peptide is derived from the 68-kDa peptide while antibodies to the 55-kDa peptide do not cross-react with other peptides found in the complex. Direct enzyme neutralization studies demonstrated that antibodies to 185- and 160-kDa peptides inhibit DNA polymerase activity in the complex, confirming earlier suggestions that these peptides are the catalytic peptides for DNA polymerase. DNA primase activity in the complex is inhibited by antibodies to 68-, 55-, and 48-kDa peptides and to a lesser extent by antibodies to the 160-kDa peptide. Free DNA primase isolated from the complex was estimated to have a native molecular weight of about 110,000. The 55- and 48-kDa peptides are found to be associated with the free primase activity. Rabbit antibodies to both 55- and 48-kDa peptides are inhibitory to this primase activity. From these results we suggest that the native calf thymus DNA polymerase-DNA primase complex contains only three unique peptides with the 185-kDa peptide as the catalytic peptide of DNA polymerase and the 55- and 68-kDa peptides constituting the primase peptides. A model illustrating the roles of these peptides in initiation and replication of DNA is presented.     

Structural and enzymological characterization of immunoaffinity-purified DNA polymerase alpha.DNA primase complex from KB cells

We describe the polypeptide structure and some of the catalytic properties of a DNA polymerase alpha.DNA primase complex that can be prepared from KB cells by immunoaffinity purification. The procedure is based on monoclonal antibodies that were raised against a biochemically purified, catalytically active core protomer of the polymerase. In all respects tested, the basic mechanism of substrate recognition and binding by the immunoaffinity-purified polymerase is qualitatively identical to that of the core protomer. The immunoaffinity-purified KB cell polymerase alpha X DNA primase is structurally complex. On the basis of extensive immunochemical analyses with five independent monoclonal antibodies, three of which are potent neutralizers of polymerase alpha activity, peptide mapping studies, and the application of a sensitive immunoassay that permits detection of polymerase alpha antigens in crude cell lysates, we have established that the principal form of catalytically active DNA polymerase alpha in KB cells is a phosphoprotein with a molecular mass of 180 kilodaltons. This protein is stable in vivo, with an estimated half-life of greater than or equal to 15 h. In contrast, the polypeptide is extremely fragile in vitro and generates partial degradation products of p165, p140, and p125 that explain the "microheterogeneity" typically exhibited by polymerase alpha peptides in denaturing polyacrylamide gels. In addition to the catalytically active polymerase alpha polypeptide(s), the immunopurified enzyme fraction typically contains three other proteins, p77, p55, and p49, the functions of which have not yet been established. These proteins do not display polymerase alpha epitopes and have been shown by peptide mapping to be independent species that are unrelated either to the large polymerase peptides or to one another. The polypeptide p77 is also a phosphoprotein, and in both p180 and p77 the phosphorylated amino acids are exclusively serine and threonine.     

The evolutionary conservation of DNA polymerase alpha.

The evolutionary conservation of DNA polymerase alpha was assessed by immunological and molecular genetic approaches. Four anti-human KB cell DNA polymerase alpha monoclonal antibodies were tested for their ability to recognize a phylogenetically broad array of eukaryotic DNA polymerases. While the single non-neutralizing antibody used in this study recognizes higher mammalian (human, simian, canine, and bovine) polymerases only, three neutralizing antibodies exhibit greater, but variable, extents of cross-reactivity among vertebrate species. The most highly cross-reactive antibody recognizes a unique epitope on a 165-180 kDa catalytic polypeptide in cell lysates from several eukaryotic sources, as distant from man as the amphibians. Genomic Southern hybridization studies with the cDNA of the human DNA polymerase alpha catalytic polypeptide identify the existence of many consensus DNA sequences within the DNA polymerase genes of vertebrate, invertebrate, plant and unicellular organisms. These findings illustrate the differential evolutionary conservation of four unique epitopes on DNA polymerase alpha among vertebrates and the conservation of specific genetic sequences, presumably reflective of critical functional domains, in the DNA polymerase genes from a broad diversity of living forms.     

The fidelity of DNA synthesis by the catalytic subunit of yeast DNA polymerase alpha alone and with accessory proteins

The fidelity of DNA synthesis catalyzed by the 180-kDa catalytic subunit (p180) of DNA polymerase alpha from Saccharomyces cerevisiae has been determined. Despite the presence of a 3'----5' exonuclease activity (Brooke et al., 1991, J. Biol. Chem., 266, 3005-3015), its accuracy is similar to several exonuclease-deficient DNA polymerases and much lower than other DNA polymerases that have associated exonucleolytic proofreading activity. Average error rates are 1/9900 and 1/12,000, respectively, for single base-substitution and minus-one nucleotide frameshift errors; the polymerase generates deletions as well. Similar error rates are observed with reactions containing the 180-kDa subunit plus an 86-kDa subunit (p86), or with these two polypeptides plus two additional subunits (p58 and p49) comprising the DNA primase activity required for DNA replication. Finally, addition of yeast replication factor-A (RF-A), a protein preparation that stimulates DNA synthesis and has single-stranded DNA-binding activity, yields a polymerization reaction with 7 polypeptides required for replication, yet fidelity remains low relative to error rates for semiconservative replication. The data suggest that neither exonucleolytic proofreading activity, the beta subunit, the DNA primase subunits nor RF-A contributes substantially to base substitution or frameshift error discrimination by the DNA polymerase alpha catalytic subunit.