The 68 kDa calmodulin-binding protein is tightly associated with the multiprotein DNA polymerase alpha-primase complex in HeLa cells.

Calcium and its receptor protein calmodulin function in the regulation of proliferation of mammalian cells. A 68 kDa calmodulin-specific binding protein was shown previously to be associated with growth factor-dependent progression of a variety of mammalian cells from G1 to S phase and to stimulate DNA synthesis in permeabilized hematopoietic progenitor cells. In this report we show that the 68 kDa calmodulin-specific binding protein in HeLa cells is tightly associated with the DNA polymerase alpha-primase component of the 21S complex of enzymes for DNA synthesis. The 68 kDa calmodulin-binding protein and the DNA polymerase alpha-primase complex cofractionate during Q-Sepharose chromatography to isolate the 21S enzyme complex, native and denatured DNA-cellulose to dissociate the 21S complex, and DEAE-Bio-Gel chromatography to isolate the multiprotein DNA polymerase alpha-primase complex. The 68 kDa calmodulin-specific binding protein and DNA polymerase alpha also bind and coelute during affinity chromatography on calmodulin-agarose. They also coprecipitate with C10-agarose-linked monoclonal antibody SJK 132-20 to human DNA polymerase alpha. The tight association of the 68 kDa calmodulin-binding protein to the DNA polymerase alpha-primase complex supports a function for this protein in the regulation of DNA synthesis in vivo.

DNA-dependent ATPase from HeLa cells is related to human Ku autoantigen.

A 150-kDa DNA-dependent ATPase composed of 83/68-kDa subunits was previously reported to cofractionate with a 21S complex of enzymes for DNA synthesis from HeLa cells (Vishwanatha, J. K., & Baril, E. F. (1990) Biochemistry 29, 8753-8759). The DNA-dependent ATPase was purified to electrophoretic homogeneity from a HeLa cell homogenate by a modified procedure that involves subcellular fractionation, poly(ethylene-glycol) precipitation of the combined nuclear extract/cytosol, and chromatography on Q-Sepharose and native and denatured DNA/celluloses followed by Mono-S fast protein liquid chromatography. The purified enzyme showed equimolar amounts of 83- and 68-kDa polypeptides following polyacrylamide gel electrophoresis under denaturing conditions. Sequence analysis of peptide fragments derived from the separated 83- and 68-kDa polypeptides showed 90-100% homology with the corresponding 80- and 70-kDa subunits of human Ku protein. Immunoblot analysis of the ATPase during the course of its purification and immunoprecipitation with antibodies to the 80- and 70-kDa subunits of human Ku protein confirmed the relationship of the 83- and 68-kDa polypeptides of the human DNA-dependent ATPase to the subunits of human Ku protein. Both the 83- and 68-kDa polypeptides are phosphorylated by a DNA-dependent protein kinase that cofractionates with the ATPase. The DNA-dependent ATPase activity is up regulated by phosphorylation.

DNA ligase I is associated with the 21 S complex of enzymes for DNA synthesis in HeLa cells.

Approximately 80% of the DNA ligase activity in HeLa cell extracts is associated with the 21 S enzyme complex that functions in simian virus 40 DNA replication in vitro (Malkas et al., Biochemistry 29, 6362-6374., 1990). The DNA ligase associated with the 21 S complex was purified extensively and its physical, enzymic and immunological properties characterized as DNA ligase I. The association of DNA ligase I with the 21 S complex of enzymes for DNA synthesis provides evidence for the physiological function of this DNA ligase in DNA replication in human cells.

Further purification and characterization of a multienzyme complex for DNA synthesis in human cells.

The 21 S complex of enzymes for DNA synthesis in the combined low salt nuclear extract-post microsomal supernatant from HeLa cells [Malkas et al. (1990) Biochemistry 29:6362-6374] was purified by poly (ethylene glycol) precipitation, Q-Sepharose chromatography, Mono Q Fast Protein Liquid Chromatography (FPLC), and velocity gradient centrifugation. The procedure gives purified enzyme complex at a yield of 45%. The 21 S enzyme complex remains intact and functional in the replication of simian virus 40 DNA throughout the purification. Sedimentation analysis showed that the 21 S enzyme complex exists in the crude HeLa cell extract and that simian virus 40 in vitro DNA replication activity in the cell extract resides exclusively with the 21 S complex. The results of enzyme and immunological analysis indicate that DNA polymerase alpha-primase, a 3',5' exonuclease, DNA ligase I, RNase H, and topoisomerase I are associated with the purified enzyme complex. Denaturing polyacrylamide gel electrophoresis of the purified enzyme complex showed the presence of about 30 polypeptides in the size range of 300 to 15 kDa. Immunofluorescent imaging analysis, with antibodies to DNA polymerase alpha,beta and DNA ligase I, showed that polymerase alpha and DNA ligase I are localized to granular-like foci within the nucleus during S-phase. In contrast, DNA polymerase beta, which is not associated with the 21 S complex, is diffusely distributed throughout the nucleoplasm.

Single-stranded-DNA-dependent ATPase from HeLa cells that stimulates DNA polymerase alpha-primase activity: purification and characterization of the ATPase.

A single-stranded DNA-dependent ATPase that cofractionates during the early stages of purification of a multiprotein DNA polymerase alpha complex from HeLa cells has been purified to homogeneity. The ATPase is part of a 16S multienzyme DNA polymerase alpha complex that is fully active in SV40 DNA replication in vitro. The ATPase hydrolyzes ATP to ADP in a reaction that is completely dependent on the presence of DNA. DNA in single-stranded form is strongly preferred as a cofactor, and polydeoxynucleotides with adenine or thymidine residues are highly effective. Glycerol gradient sedimentation showed that the purified ATPase sedimented at an s20,w of 7 S, and polyacrylamide gel electrophoresis under denaturing conditions reveals two polypeptides with relative molecular weights of 83,000 and 68,000. Both of these polypeptides have purine nucleotide binding sites as revealed by photoaffinity cross-linking experiments. ATP binds to the two subunits more efficiently than GTP, and CTP or UTP does not cross-link with the two polypeptides. DNA synthesis catalyzed by purified HeLa cell DNA polymerase alpha-primase is stimulated in the presence of ATPase and ATP at an optimum concentration of 2 mM. Analysis of the DNA product by gel electrophoresis indicates that with poly(dT) but not phage M13 DNA as template the ATPase overcomes a lag and decreases the length of nascent DNA chains synthesized by the DNA polymerase alpha-primase complex.

A 21S enzyme complex from HeLa cells that functions in simian virus 40 DNA replication in vitro.

A sedimentable complex of enzymes for DNA synthesis was partially purified from the combined low-salt nuclear extract-postmicrosomal supernatant solution of HeLa cell homogenates by poly(ethylene glycol) precipitation in the presence of 2 M KCl, discontinuous gradient centrifugation, Q-Sepharose chromatography, and velocity gradient centrifugation. In addition to the previously described 640-kDa multiprotein DNA polymerase alpha-primase complex [Vishwanatha et al. (1986) J. Biol. Chem. 261, 6619-6628], the enzyme complex also has associated topoisomerase I, DNA-dependent ATPase, RNase H, DNA ligase, a simian virus 40 origin recognition, dA/dT sequence binding protein [Malkas & Baril (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 70-74], and proliferating cell nuclear antigen. Essentially all of the T antigen dependent simian virus 40 in vitro replication activity in the combined nuclear extract-postmicrosomal supernatant solution resides with the sedimentable complex of enzymes for DNA synthesis. Sedimentation analysis on a 10-35% glycerol gradient in the presence of 0.5 M KCl indicates that the enzyme complex is 21S. The associated enzymes for DNA synthesis and in vitro simian virus 40 replication activity cofractionate throughout the purification of the 21S complex. The DNA polymerase and in vitro simian virus 40 replication activities are both inhibited by monoclonal antibody (SJK 132-20) to human DNA polymerase alpha and by 5-10 microM butylphenyl-dGTP, indicating that the association of DNA polymerase alpha with the 21S enzyme complex is essential for the initiation of SV40 DNA replication in vitro.

Sequence recognition protein for the 17-base-pair A + T-rich tract in the replication origin of simian virus 40 DNA.

A DNA-binding protein has been identified that recognizes runs of deoxyadenines and/or deoxythymines (dA/dT sequences) and purified from a chromatographic fraction containing the multiprotein DNA polymerase alpha-primase complex of HeLa cells by successive steps of chromatography on oligo(dT)-cellulose and Q-Sepharose. Polyacrylamide gel electrophoresis of the purified dA/dT sequence-binding protein in the presence of NaDodSO4 showed a single protein band of 62 kDa. Nitrocellulose filter binding assays using homopolydeoxynucleotides indicated that the purified protein preferentially binds to dA/dT sequences in single-stranded or duplex DNAs. Gel mobility shift assays with a variety of DNAs showed that the purified protein specifically binds to a fragment of simian virus 40 DNA containing the minimal (core) origin for replication. The binding occurred in a protein-dependent manner and in the presence of a vast excess of competing DNAs lacking the simian virus replication origin. The origin binding was reduced, however, when DNA fragments from simian virus 40 deletion mutants containing deletions within the 17-base-pair A + T-rich tract in the core DNA replication origin were used in the assays. These results indicate that the dA/dT sequence-binding protein preferentially binds to the 17-base-pair A + T-rich tract and suggest a possible role for the protein in the initiation of DNA replication.

Resolution and purification of free primase activity from the DNA primase-polymerase alpha complex of HeLa cells.

DNA primase activity has been resolved from a purified DNA primase-polymerase alpha complex of HeLa cells by hydrophobic affinity chromatography on phenylSepharose followed by chromatography on hexylagarose. This procedure provides a good yield (55%) of DNA primase that is free from polymerase alpha. The free DNA primase activity was purified to near homogeneity and its properties characterized. Sodium dodecyl sulfate polyacrylamide gel electrophoretic analysis of the purified free DNA primase showed a major protein staining band of Mr 70,000. The native enzyme in velocity sedimentation has an S20'W of 5. DNA primase synthesizes RNA oligomers with single-stranded M-13 DNA, poly(dT) and poly(dC) templates that are elongated by the DNA polymerase alpha in a manner that has already been described for several purified eukaryotic DNA primase-polymerase alpha complexes. The purified free DNA primase activity is resistant to neutralizing anti-human DNA polymerase alpha antibodies, BuPdGTP and aphidicolin that specifically inhibit the free DNA polymerase alpha and also DNA polymerase alpha complexed with the primase. The free primase activity is more sensitive to monovalent salt concentrations and is more labile than polymerase alpha. Taken together these results indicate that the DNA primase and polymerase alpha activities of the DNA primase-polymerase alpha complex reside on separate polypeptides that associate tightly through hydrophobic interactions.

Selection of template initiation sites and the lengths of RNA primers synthesized by DNA primase are strongly affected by its organization in a multiprotein DNA polymerase alpha complex.

Synthesis of (p)ppRNA-DNA chains by purified HeLa cell DNA primase-DNA polymerase alpha (pol alpha-primase) was compared with those synthesized by a multiprotein form of DNA polymerase alpha (pol alpha 2) using unique single-stranded DNA templates containing the origin of replication for simian virus 40 (SV40) DNA. The nucleotide locations of 33 initiation sites were identified by mapping G*pppN-RNA-DNA chains and identifying their 5'-terminal ribonucleotide. Pol alpha 2 strongly preferred initiation sites that began with ATP rather than GTP, thus frequently preferring different initiation sites than pol alpha-primase, depending on the template examined. The initiation sites selected in vitro, however, did not correspond to the sites used during SV40 DNA replication in vivo. Pol alpha 2 had the greatest effect on RNA primer size, typically synthesizing primers 1-5 nucleotides long, while pol alpha-primase synthesized primers 6-8 nucleotides long. These differences were observed even at individual initiation sites. Thus, the multiprotein form of DNA primase-DNA polymerase alpha affects selection of initiation sites, the frequency at which the sites are chosen, and length of RNA primers.

A multiprotein form of DNA polymerase alpha from HeLa cells. Resolution of its associated catalytic activities.

The majority of the DNA polymerase alpha activity in HeLa cells has been isolated and purified as a multiprotein Mr 640,000 form. The multiprotein form of DNA polymerase alpha corresponds to DNA polymerase alpha 2 that was previously reported by us (Lamothe, P., Baril, B., Chi, A., Lee, L., and Baril, E. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 4723-4727). The highly purified DNA polymerase alpha 2 has in addition to DNA polymerase alpha-associated DNase, primase, and diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A)binding activities and accessory primer recognition proteins C1 and C2. The DNA polymerase alpha and associated activities increase coordinately during the G1/S-phase transition of the cell cycle. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the electrophoretically homogeneous DNA polymerase alpha shows that it is composed of at least eight polypeptides in the molecular weight range of 180,000-15,000. Hydrophobic chromatography on butyl-agarose resolves the DNase and Ap4A-binding protein from a complex of DNA polymerase alpha, primase, and the primer recognition proteins C1 and C2. Hydrophobic chromatography of the latter complex on phenyl-Sepharose resolves the C1 protein from a DNA polymerase alpha-C2 protein-primase complex. Phosphocellulose chromatography of the DNA polymerase-primase-C2 protein complex resolves the C2 protein from a complex of DNA polymerase alpha-primase.

5',5'''-P1, P4 diadenosine tetraphosphate (Ap4A): a putative initiator of DNA replication.

The proposal that Ap4A acts as an inducer of DNA replication is based primarily on two pieces of evidence (7). The intracellular levels of Ap4A increase ten- to 1000-fold as cells progress into S phase and the introduction of Ap4A into nonproliferating cells stimulated DNA synthesis. There is also some additional suggestive evidence such as the binding of Ap4A to a protein that is associated with multiprotein forms of the replicative DNA polymerase alpha and the ability of this enzyme to use Ap4A as a primer for DNA synthesis in vitro with single-stranded DNA templates. These observations have stimulated interest in the cellular metabolism of Ap4A. This is well since there is a great need for additional experimentation in order to clearly establish Ap4A as an inducer of DNA replication. Microinjection experiments of Ap4A into quiescent cells are needed in order to ascertain if Ap4A will stimulate DNA replication and possibly cell division in intact cells. Studies of the effects of nonhydrolyzable analogs of Ap4A on DNA replication in intact quiescent cells could also prove valuable. Although Ap4A can function as a primer for in vitro DNA synthesis by DNA polymerase alpha this may not be relevant in regard to its in vivo role in DNA replication. Ap4A in vivo could interact with key protein(s) in DNA replication and in this way act as an effector molecule in the initiation of DNA replication. In this regard the interaction of Ap4A with a protein associated with a multiprotein form of DNA polymerase alpha isolated from S-phase cells is of interest. More experiments are required to determine if there is a specific target protein(s) for Ap4A in vivo and what its role in DNA replication is. The cofractionation of tryptophanyl-tRNA synthetase with the replicative DNA polymerase alpha from animal and plant cells is of interest. The DNA polymerase alpha from synchronized animal cells also interacted with Ap4A. Although the plant cell alpha-like DNA polymerase did not interact with Ap4A this DNA polymerase was not a multiprotein form of polymerase alpha and the synchrony of the wheat germ embryos was not known. A possible tie between protein-synthesizing systems and the regulation of proteins involved in DNA replication may exist. The requirement of protein synthesis for the initiation of DNA replication has long been known. Also, it is well established that many temperature-sensitive mutants for tRNA synthetases are also DNA-synthesizing mutants. More investigation in this area may be warranted.(ABSTRACT TRUNCATED AT 400 WORDS)

DNA polymerase alpha cofactors C1C2 function as primer recognition proteins.

Most, if not all, of the DNA polymerase alpha activity in monkey and human cells was complexed with at least two proteins, C1 and C2, that together stimulated the activity of this enzyme from 180- to 1800-fold on low concentrations of denatured DNA, parvovirus DNA, M13, and phi X174 DNA or RNA-primed DNA templates, and poly(dT):oligo(dA) or oligo(rA). These primer-template combinations, which have from 200 to 5000 bases of template/primer, were then 7- to 50-fold more effective as substrates than DNase I-activated DNA. C1C2 specifically stimulated alpha polymerase, and only from the same cell type. Alpha X C1C2-polymerase reconstituted from purified alpha polymerase and the C1C2 cofactor complex behaved the same as native alpha X C1C2-polymerase and C1C2 had no effect on the sensitivity of alpha polymerase to aphidicolin, dideoxythymidine triphosphate, and N-ethylmaleimide. In the presence of substrates with a high ratio of single-stranded DNA template to either DNA or RNA primar, C1C2 increased the rate of DNA synthesis by decreasing the Km for the DNA substrate, decreasing the Km for the primer itself, increasing the use of shorter primers, and stimulating incorporation of the first deoxyribonucleotide. In contrast, C1C2 had no effect on the Km values for deoxyribonucleotide substrates (which were about 150-fold higher than for DNA replication in isolated nuclei), the ability of specific DNA sequences to arrest alpha polymerase, or the processivity of alpha polymerase. Accordingly, C1C2 function as primer recognition proteins. However, C1C2 did not reduce the comparatively high Km values or stimulate DNA synthesis by alpha polymerase on lambda DNA ends and DNase I-activated DNA, substrates with 12 and about 30-70 bases of template/primer, respectively. DNA restriction fragments with 1 to 4 bases of template/primer were substrates for neither alpha nor alpha X C1C2-polymerase. Therefore, we propose that C1C2 enhances the ability of alpha polymerase to initiate DNA synthesis by eliminating nonproductive binding of the enzyme to single-stranded DNA, allowing it to slide along the template until it recognizes a primer.

Design and characterization of N2-arylaminopurines which selectively inhibit replicative DNA synthesis and replication-specific DNA polymerases: guanine derivatives active on mammalian DNA polymerase alpha and bacterial DNA polymerase III.

The 2-amino substituted derivatives of guanine, N2-(p-n-butylphenyl)guanine (BuPG) and N2-(3',4'-trimethylenephenyl) guanine (TMPG), were synthesized and found to selectively inhibit, respectively, HeLa cell DNA polymerase alpha (po1 alpha) and B. subtilis DNA polymerase III (po1 III). Both purines, like their corresponding uracil analogs, BuAu and TMAU (2,9), were specifically competitive with dGTP in their inhibitory action on their target polymerases. BuPG, the pol alpha-specific purine, was also toxic for HeLa cells in vivo, selectively inhibiting DNA synthesis. These N2-substituted purines, in contrast to the 6-substituted uracils, provide a structural basis for the synthesis of nucleosides and nucleotides with considerable potential as probes for the analysis of the structure of specific replicative DNA polymerases and their function in cellular DNA metabolism.

Priming of DNA synthesis by diadenosine 5',5"'-P1,P4-tetraphosphate with a double-stranded octadecamer as a template and DNA polymerase alpha.

Diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) primes DNA synthesis in an in vitro system containing purified HeLa cell DNA polymerase alpha, deoxyadenosine triphosphate, and the double-stranded synthetic octadecamer template 5'-d-(G-G-A-G-G-C-T-T-T-T-T-T-G-G-A-G-G-C) (C-C-T-C-C-G-A-A-A-A-A-A-C-C-T-C-C-G)-d-5'; this octadecamer sequence is part of the origin region of DNA synthesis in simian virus 40. Ap4A is shown to be covalently linked to the first residue of the short deoxynucleotide chain synthesized under these experimental conditions. This template-primer system can initiate the new deoxynucleotide chain but cannot extend it beyond the A . T region.

Association of diadenosine 5',5"'-P1,P4-tetraphosphate binding protein with HeLa cell DNA polymerase alpha.

An electrophoretically homogeneous high molecular weight form (640,000) of HeLa cell DNA polymerase alpha was shown to catalyze DNA synthesis with a variety of di- and oligoriboadenylates and oligodeoxyriboadenylates as primers with poly(dT) as template. Diadenosine 5',5"'-P1,p4-tetraphosphate (Ap4A) can be utilized as a primer with poly(dT) as template and was found to be covalently attached to the 5'-end of the poly(dA) product. An Ap4A binding protein is tightly associated with the high molecular weight form of DNA polymerase alpha. This protein which exhibits high affinity, noncovalent binding of Ap4A is resolved from the multiprotein DNA polymerase alpha complex, along with other accessory proteins, by hydrophobic affinity chromatography on phenyl-Sepharose columns.

HeLa cell DNA polymerase alpha is tightly associated with tryptophanyl-tRNA synthetase and diadenosine 5',5"'-P1,P4-tetraphosphate binding activities.

The purified high molecular weight form of HeLa cell DNA polymerase alpha (deoxynucleosidetriphosphate: DNA deoxynucleotidyltransferase, EC 2.7.7.7) was shown to associate tightly with several aminoacyl-tRNA synthetase activities. Fractionation of the high molecular weight enzyme on hexylagarose followed by gel filtration, chromatography on phosphocellulose, or polyacrylamide gel electrophoresis under nondenaturing conditions demonstrated copurification of only tryptophanyl-tRNA synthetase [L-tryptophan:tRNATrp ligase (AMP-forming), EC 6.1.1.2] along with DNA polymerase alpha. The high molecular weight (660,000) and low molecular weight (145,000) forms of DNA polymerase alpha were shown to possess a highly specific, noncovalent, diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) binding activity. The dissociation constants were determined to be 16 and 22 microM, respectively, by utilization of a charcoal adsorption procedure. No high-affinity binding of ATP could be detected. These findings suggest a link between the amino acid activation process and DNA replication in mammalian cells.

A beta-like DNA polymerase activity in the slime mold Dictyostelium discoideum.

Two distinct DNA polymerases (DNA nucleotidyltransferases) have been partially purified from exponentially growing cells of the slime mold Dictyostelium discoideum. The major DNA polymerase activity, DNA polymerase A, has physical and enzymatic properties similar to DNA polymerase alpha from vertebrate cells. DNA polymerase B, representing less than 10% of the polymerase activity in homogenates, is similar to DNA polymerase beta from vertebrate cells in that its activity is not inhibited by N-ethylmaleimide, aphidicolin, or KCl at a concentration below 200 mM. Like DNA polymerase beta, the activity of Dictyostelium DNA polymerase B is sensitive to 2', 3'-dideoxythymidine 5'-triphosphate. However, it differs from vertebrate DNA polymerase beta in that it chromatographs as an acidic protein and has an apparent molecular weight of 70,000. Previous reports of the absence of a beta-like DNA polymerase in D. discoideum apparently resulted from inadequate extraction and assay conditions for the enzyme. It seems quite probable from the results of this study and those of other studies (e.g., yeast and Tetrahymena pyriformis) that lower eukaryotic organisms, as well as vertebrates and prokaryotes, contain more than one form of DNA polymerase.

Butylanilinouracil: a selective inhibitor of HeLa cell DNA synthesis and HeLa cell DNA polymerase alpha.

A series of 6-anilinouracils, dGTP analogues which selectively inhibit specific bacterial DNA polymerases, were examined for their capacity to inhibit purified DNA polymerases from HeLa cells. The p-n-butyl derivative (BuAU) was found to inhibit DNA polymerase alpha with a Ki of approximately 60 microM. The inhibitory effect of BuAU was reversed specifically by dGTP and was observed only for DNA polymerase alpha; polymerases beta and lambda were not inhibited by drug at concentrations as high as 1 mM. BuAU also was inhibitory in vivo in HeLa cell culture; at 100 microM it reversibly inhibited cell division and selectively depressed DNA synthesis. The results of these studies indicate that BuAU is an inhibitor with considerable potential as a specific probe with which to dissect the structure of mammalian polymerase alpha and its putative role in cellular DNA replication.

HeLa DNA polymerase alpha activity in vitro: specific stimulation by a non-enzymic protein factor.

A non-enzymic protein factor that increases the in vitro rate of synthesis by HeLa DNA polymerase alpha 15- to 30-fold with denatured DNA as template has been partially purified from the cytoplasmic fraction of HeLa cells. The stimulatory effect is highly specific for HeLa DNA polymerase alpha and for DNA templates that contain extensive regions of single-strandedness. Synthesis with denatured DNA as template presumably proceeds from 3'-hydroxyl termini formed at loop-back regions since the synthesized DNA product and template are covalently linked. The stimulatory protein factor chromatographs as a basic protein, has an approximate molecular weight of 30,000 daltons and binds with moderate affinity to denatured DNA cellulose, being eluted by o.4M NaCl. The purified factor lacks detectable DNA polymerase, exo- and endodeoxyribonuclease and RNA polymerase activities. It also does not promote helix-coil transitions with poly[d(A-T)] and Clostridium perfringens DNA.

Induction of DNA polymerase alpha during liver regeneration in rats on controlled feeding schedules.

The activity of 2 nonmitochondrial forms of DNA polymerase, designated DNA polymerases alpha and beta, was investigated during liver regeneration in regimented rats. In accord with Barbiroli and Potter, we observed that regimentation of rats with respect to temperature, light and darkness, and availability of food resolves the DNA synthesis response to partial hepatectomy into 2 peaks, one occurring at a fixed time after operation and the other entrained by the environmental conditions. The peaks can be fused or separated depending on the timing of the operation. For this study, operation times were selected to give both patterns of DNA synthesis as measured by the uptake of radioactive thymidine into DNA. For both operation times, DNA polymerase activity in the nuclear extract correlated temporally and qualitatively with radioactive thymidine uptake into DNA. At the times of maximal DNA synthesis and polymerase activity, the DNA polymerase was purified from extracts of isolated nuclei. DNA polymerase alpha represented 70% and DNA polymerase beta represented 30% of the recovered activity from the nuclear extract. This is in agreement with the previous observation in nonregimented rats that DNA polymerase alpha is the major activity in nuclei during liver regeneration. For both operation times, DNA polymerase activity in the postmicrosomal fraction was sedimentable and increased 3 to 4 times above the level observed with this same fraction from normal rat liver. This activity was shown to be due to DNA polymerase alpha only with this subcellular fraction. DNA polymerase alpha activity with this fraction peaked 4 to 6 hr after the time of maximal radioactive thymidine incorporation into DNA. DNA polymerase activity in the microsome fraction did not change significantly after partial hepatectomy. This activity has been shown to represent DNA polymerase beta. Prior administration of cycloheximide and actinomycin abolished the rise in DNA polymerase alpha activity in the nucleus and postmicrosomal fraction. Hydroxyurea did not prevent the rise in DNA polymerase alpha activity with those subcellular fractions but did inhibit over 90% of the uptake of radioactive thymidine into DNA. These data suggest, but do not prove, that DNA polymerase alpha activity is induced in response to the stimulus(i) for liver regeneration.