Evidence that a high molecular weight replicative DNA polymerase is conserved during evolution.

Using a technique developed recently to detect DNA polymerase activity in situ after NaDodSO4 gel electrophoresis (Spanos, A., Sedgwick, S. G., Yarranton, g. T., Hübscher, U. & Banks, G. R. (1981) Nucleic Acids Res. 9, 1825-1839), we present evidence that a high Mr (greater than or equal to 125,000) polypeptide is responsible for chromosomal DNA replication in prokaryotes, lower eukaryotes and high eukaryotes. Not only extracts from Escherichia coli, Ustilago maydis, Drosophila melanogaster, rat neurones, calf thymus, human fibroblast, and HeLa cells possess such high Mr activities, but also highly purified E. coli DNA polymerase III core enzyme, U. maydis DNA polymerase, and D. melanogaster embryo and calf thymus DNA alpha polymerases. The evidence that these activities are responsible for chromosomal DNA replication is genetical (E. coli, U. maydis, and D. melanogaster); also, the high Mr activity disappears from rat neurones during differentiation from an actively dividing precursor cell to a postmitotically mature neurone. Furthermore, when limited proteolysis is allowed to occur, a defined and remarkably similar pattern of intermediate Mr activities is generated in lower eukaryotic and high eukaryotic extracts and, to some extent, in prokaryotic extracts. In higher eukaryotic extracts, a low Mr activity of approximately 35,000 is also generated. Protease inhibitors can retard formation of these catalytically active proteolytic fragments. We propose that the replicative DNA polymerase complex of both prokaryotes and eukaryotes contains a high Mr polypeptide responsible for chain elongation which might be conserved during evolution and which is extremely sensitive to proteolytic cleavage.     

A 5'' to 3'' exonuclease functionally interacts with calf DNA polymerase epsilon.

Analysis of fractions containing purified DNA polymerase epsilon from calf thymus has revealed the presence of a 5' to 3' exonuclease activity that is specific for a single strand of duplex DNA. This activity is capable of degrading a 3'-labeled oligonucleotide hybridized to M13mp18 DNA. When a second oligonucleotide primer is annealed 3 bases upstream, degradation of the downstream primer is strictly dependent on DNA synthesis from the upstream primer. Replacement of the downstream primer by an oligoribonucleotide of identical sequence results in a similar pattern of exonucleolytic activity. The activity has been highly purified and found to cosediment in glycerol gradients with a peptide of 56 kDa as judged by SDS/PAGE analysis. Effects of calf DNA polymerase alpha and delta on exonuclease activity are also observed but with differences in the pattern of products.     

Association of proliferating cell nuclear antigen with cyclin-dependent kinases and cyclins in normal and transformed human T lymphocytes

The proliferating cell nuclear antigen (PCNA) is an auxiliary protein of DNA polymerase delta and appears to be needed for both DNA synthesis and DNA repair. It is present in low amount in resting normal human T lymphocytes and, upon mitogenic stimulation with phorbol dibutyrate and ionomycin, begins to increase in mid-G1 phase, approximately 12 to 15 hours before entry into S phase. PCNA continues to increase in amount throughout the cell cycle and remains high in proliferating cultures. PCNA was extracted from activated normal T cells and from the transformed T-lymphoblastoid cell line Jurkat by a method that recovered approximately 98% of total cellular PCNA but yet retained its associations with other proteins. PCNA immunoprecipitates possessed H1 histone kinase activity, which increased in parallel with increasing cellular content of PCNA. Both the cdc2 and cdk2 kinases were found associated with PCNA in normal T cells, in amounts consistent with detected kinase activity. The results indicate that PCNA is not an inhibitory molecule of cdk/cyclin activity. Both normal and transformed T cells contained PCNA in association with cdk2, cdk4, cdk5, and cdk6, with the amount of PCNA associated with these molecules increasing in the order listed. Relatively high amounts of PCNA were also found associated with cyclins D2 and D3, the major cyclin partners of cdk6 in T cells. Though detected in normal cells, PCNA/cdc2 complexes were present in exceedingly low amount, if at all, in Jurkat cells. This cell line appeared to contain more of nearly all of the cdk and cyclin molecules analyzed, but there seemed to be little difference in the patterns of association of these molecules with PCNA in the cell line as compared with normal human T cells.     

Cdk-interacting protein 1 directly binds with proliferating cell nuclear antigen and inhibits DNA replication catalyzed by the DNA polymerase delta holoenzyme.

Cdk-interacting protein 1 (Cip1) is a p53-regulated 21-kDa protein that inhibits several members of the cyclin-dependent kinase (CDK) family. It was initially observed in complexes containing CDK4, cyclin D, and proliferating cell nuclear antigen (PCNA). PCNA, in conjunction with activator 1, acts as a processivity factor for eukaryotic DNA polymerase (pol) delta, and these three proteins constitute the pol delta holoenzyme. In this report, we demonstrate that Cip1 can also directly inhibit DNA synthesis in vitro by binding to PCNA. Cip1 efficiently inhibits simian virus 40 replication dependent upon pol alpha, activator 1, PCNA, and pol delta, and this inhibition can be overcome by additional PCNA. Simian virus 40 DNA replication, catalyzed solely by high levels of pol alpha-primase complex, is unaffected by Cip1. Using the surface plasmon resonance technique, a direct physical interaction of PCNA and Cip1 was detected. We have observed that Cip1 efficiently inhibits synthesis of long (7.2 kb) but not short (10 nt) templates, suggesting that its association with PCNA is likely to impair the processive movement of pol delta during DNA chain elongation, as opposed to blocking assembly of the pol delta holoenzyme. The implications of the Cip1-PCNA interaction with respect to regulation of DNA synthesis, cell cycle checkpoint control, and DNA repair are discussed.     

Proliferating cell nuclear antigen promotes DNA synthesis past template lesions by mammalian DNA polymerase δ

Consistent with previous observations, proliferating cell nuclear antigen (PCNA) promotes DNA synthesis by calf thymus DNA polymerase δ (pol δ) past several chemically defined template lesions including model abasic sites, 8-oxo-deoxyguanosine (dG) and aminofluorene-dG (but not acetylaminofluorene-dG). This synthesis is potentially mutagenic. The model abasic site was studied most extensively. When all deoxyribonucleoside triphosphates and a template bearing a model abasic site were present, DNA synthesis by pol δ beyond this site was stimulated 53-fold by addition of homologous PCNA. On an unmodified template (lacking any lesions), PCNA stimulated pol δ by 1.3-fold. Product analysis demonstrated that as expected from the “A-rule,” fully and near-fully extended primers incorporated predominantly dAMP opposite the template lesion. Moreover, corollary primer extension studies demonstrated that in the presence (but not the absence) of PCNA, pol δ preferentially elongated primers containing dAMP opposite the model abasic template site. p21, a specific inhibitor of PCNA-dependent DNA replication, inhibits PCNA-stimulated synthesis past model abasic template sites. We propose that DNA synthesis past template lesions by pol δ promoted by PCNA results from the fundamental mechanism by which PCNA stimulates pol δ, i.e., stabilization of the pol δtemplate-primer complex.     

Proofreading by the epsilon subunit of Escherichia coli DNA polymerase III increases the fidelity of calf thymus DNA polymerase alpha.

Addition of the 3'----5' proofreading exonuclease, epsilon subunit of Escherichia coli DNA polymerase III, to DNA polymerase alpha from calf thymus has been studied. Alone, calf thymus DNA polymerase alpha terminates in vitro DNA synthesis upon insertion of noncomplementary nucleotides. Upon addition of the epsilon subunit, DNA polymerase alpha elongates the newly synthesized DNA as a result of hydrolysis of the 3'-terminal mispair. The fidelity of DNA polymerase alpha in vitro is increased 7-fold by addition of the exonuclease. The functional interaction between DNA polymerase alpha and the epsilon subunit is independent of any detectable physical association. This suggests that a mechanism for proofreading could exist in mammalian cells involving sequential catalysis by DNA polymerase alpha excision of errors by a separate 3'----5' exonuclease, and further elongation onto correctly base-paired 3' termini by DNA polymerase alpha.     

Mechanism of elongation of primed DNA by DNA polymerase delta, proliferating cell nuclear antigen, and activator 1.

In the presence of a single-stranded-DNA-binding protein (SSB), the elongation of primed DNA templates by DNA polymerase delta (pol delta) is dependent on ATP and two protein factors, activator 1 (A1) and proliferating cell nuclear antigen (PCNA). We have examined the interaction of these proteins with (dA)4500.(dT)12-18 by measuring their ability to form stable complexes with this DNA. In the presence of ATP, A1, PCNA, and pol delta formed a stable complex with DNA that could be isolated by gel filtration. Incubation of the isolated complex with dTTP resulted in the synthesis of poly(dT). While ATP was required for the formation of this complex, it was not required for the subsequent elongation of DNA. The temporal requirements for complex formation were determined. A1 was found to bind first, followed by the ATP-dependent addition of PCNA to the A1.DNA complex, while pol delta was added last. Each of these complexes could be isolated by gel filtration, indicating that they possessed a high degree of stability. The binding of PCNA to the A1-SSB-coated primed DNA occurred with adenosine 5'-[gamma-thio]triphosphate as well as ATP. However, the binding of pol delta to the PCNA.A1-DNA complex was observed only when the latter complex was formed in the presence of ATP. The complete complex was formed after incubation at 37 degrees C for 2 min, whereas no complex was detected after incubation at 0 degree C. These results indicate that these proteins act in a manner analogous to the accessory proteins that play critical roles in the elongation reaction catalyzed by T4 phage DNA polymerase and Escherichia coli DNA polymerase III.     

DNA polymerase beta bypasses in vitro a single d(GpG)-cisplatin adduct placed on codon 13 of the HRAS gene.

We have examined the capacity of calf thymus DNA polymerases alpha, beta, delta, and epsilon to perform in vitro translesion synthesis on a substrate containing a single d(GpG)-cisplatin adduct placed on codon 13 of the human HRAS gene. We found that DNA synthesis catalyzed by DNA polymerases alpha, delta, and epsilon was blocked at the base preceding the lesion. Addition of proliferating cell nuclear antigen to DNA polymerase delta and replication protein A to DNA polymerase alpha did not restore their capacity to elongate past the adduct. On the other hand, DNA polymerase beta efficiently bypassed the cisplatin adduct. Furthermore, we observed that DNA polymerase beta was the only polymerase capable of primer extension of a 3'-OH located opposite the base preceding the lesion. Likewise, DNA polymerase beta was able to elongate the arrested replication products of the other three DNA polymerases, thus showing its capacity to successfully compete with polymerases alpha, delta, and epsilon in the stalled replication complex. Our data suggest (i) a possible mechanism enabling DNA polymerase beta to bypass a d(GpG)-cisplatin adduct in vitro and (ii) a role for this enzyme in processing DNA damage in vivo.     

Studies on the interactions between human replication factor C and human proliferating cell nuclear antigen

Proliferating cell nuclear antigen (PCNA) is a processivity factor required for DNA polymerase delta (or epsilon)-catalyzed DNA synthesis. When loaded onto primed DNA templates by replication factor C (RFC), PCNA acts to tether the polymerase to DNA, resulting in processive DNA chain elongation. In this report, we describe the identification of two separate peptide regions of human PCNA spanning amino acids 36-55 and 196-215 that bind RFC by using the surface plasmon resonance technique. Site-directed mutagenesis of residues within these regions in human PCNA identified two specific sites that affected the biological activity of PCNA. Replacement of the aspartate 41 residue by an alanine, serine, or asparagine significantly impaired the ability of PCNA to (i) support the RFC/PCNA-dependent polymerase delta-catalyzed elongation of a singly primed DNA template; (ii) stimulate RFC-catalyzed DNA-dependent hydrolysis of ATP; (iii) be loaded onto DNA by RFC; and (iv) activate RFC-independent polymerase delta-catalyzed synthesis of poly dT. Introduction of an alanine at position 210 in place of an arginine also reduced the efficiency of PCNA in supporting RFC-dependent polymerase delta-catalyzed elongation of a singly primed DNA template. However, this mutation did not significantly alter the ability of PCNA to stimulate DNA polymerase delta in the absence of RFC but substantially lowered the efficiency of RFC-catalyzed reactions. These results are in keeping with a model in which surface exposed regions of PCNA interact with RFC and the subsequent loading of PCNA onto DNA orients the elongation complex in a manner essential for processive DNA synthesis.     

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

Proliferating-cell nuclear antigen (PCNA) mediates the replication of simian virus 40 (SV40) DNA by reversing the effects of a protein that inhibits the elongation reaction. Two other protein fractions, activator I and activator II, were also shown to play important roles in this process. We report that activator II isolated from HeLa cell extracts is a PCNA-dependent DNA polymerase delta that is required for efficient replication of DNA containing the SV40 origin of replication. PCNA-dependent DNA polymerase delta on a DNA singly primed phi X174 single-stranded circular DNA template required PCNA, a complex of the elongation inhibitor and activator I, and the single-stranded DNA-binding protein essential for SV40 DNA replication. DNA polymerase delta, in contrast to DNA polymerase alpha, hardly used RNA-primed DNA templates. These results indicate that both DNA polymerase alpha and delta are involved in SV40 DNA replication in vitro and their activity depends on PCNA, the elongation inhibitor, and activator I.     

Primary structure of the catalytic subunit of human DNA polymerase delta and chromosomal location of the gene.

The catalytic subunit (Mr approximately 124,000) of human DNA polymerase delta has been cloned by PCR using poly(A)+ RNA from HepG2 cells and primers designed from the amino acid sequence of regions highly conserved between bovine and yeast DNA polymerase delta. The human cDNA was 3443 nucleotides in length and coded for a polypeptide of 1107 amino acids. The enzyme was 94% identical to bovine DNA polymerase delta and contained the numerous highly conserved regions previously observed in the bovine and yeast enzymes. The human enzyme also contained two putative zinc-finger domains in the carboxyl end of the molecule, as well as a putative nuclear localization signal at the amino-terminal end. The gene coding for human DNA polymerase delta was localized to chromosome 19.     

Functional interaction between the Werner Syndrome protein and DNA polymerase delta

Werner Syndrome (WS) is an inherited disease characterized by premature onset of aging, increased cancer incidence, and genomic instability. The WS gene encodes a 1,432-amino acid polypeptide (WRN) with a central domain homologous to the RecQ family of DNA helicases. Purified WRN unwinds DNA with 3'-->5' polarity, and also possesses 3'-->5' exonuclease activity. Elucidation of the physiologic function(s) of WRN may be aided by the identification of WRN-interacting proteins. We show here that WRN functionally interacts with DNA polymerase delta (pol delta), a eukaryotic polymerase required for DNA replication and DNA repair. WRN increases the rate of nucleotide incorporation by pol delta in the absence of proliferating cell nuclear antigen (PCNA) but does not stimulate the activity of eukaryotic DNA polymerases alpha or epsilon, or a variety of other DNA polymerases. Moreover, we show that functional interaction with WRN is mediated through the third subunit of pol delta: i.e., Pol32p of Saccharomyces cerevisae, corresponding to the recently identified p66 subunit of human pol delta. Absence of the third subunit abrogates stimulation by WRN, and stimulation is restored by reconstituting the three-subunit enzyme. Our findings suggest that WRN may facilitate pol delta-mediated DNA replication and/or DNA repair and that disruption of WRN-pol delta interaction in WS cells may contribute to the previously observed S-phase defects and/or the unusual sensitivity to a limited number of DNA damaging agents.     

Sequence and expression in Escherichia coli of the 40-kDa subunit of activator 1 (replication factor C) of HeLa cells.

Activator 1 (A1; also called replication factor C), in conjunction with proliferating-cell nuclear antigen (PCNA), is essential for the elongation of primed DNA templates by DNA polymerases delta and epsilon. A1 contains five distinct subunits of 145, 40, 38, 37, and 36.5 kDa. Here we describe the isolation, sequence, and bacterial expression of a cDNA coding for the 40-kDa subunit. In keeping with the presence of an ATP-binding motif, the bacterially expressed 40-kDa subunit binds ATP. The interaction between the 40-kDa subunit and ATP was reduced by the addition of PCNA. In addition, antibodies raised against the 40-kDa subunit abolished the A1- and PCNA-dependent synthesis of DNA catalyzed by polymerase delta. The putative amino acid sequence of the 40-kDa subunit of A1 revealed significantly homology with the bacteriophage T4 gene 44 protein and, to a lesser degree, with the tau and gamma subunits of Escherichia coli DNA polymerase III holoenzyme.     

Exonucleolytic proofreading by calf thymus DNA polymerase delta.

The fidelity of DNA synthesis by calf thymus DNA polymerase delta (pol delta) in vitro has been determined using an M13lacZ alpha nonsense codon reversion assay. Pol delta is highly accurate, producing on average less than 1 single-base substitution error for each 10(6) nucleotides polymerized. This accuracy is 10- and 500-fold greater than that of DNA polymerases alpha and beta, respectively, in the same assay. Three observations suggest that this higher fidelity results in part from proofreading of misinserted bases by the 3' to 5' exonuclease associated with pol delta. First, the exonuclease efficiently excises terminally mismatched bases. Second, both terminal mismatch excision and the fidelity of DNA synthesis by pol delta are reduced with increasing concentration of deoxynucleoside triphosphates in the synthesis reaction. These effects result from increasing the rate of polymerization relative to the rate of exonucleolytic excision and are hallmarks of exonuclease proofreading. Third, both terminal mismatch excision and fidelity decrease upon addition to the reaction mixture of adenosine monophosphate, a compound known to selectively inhibit the exonuclease but not the polymerase activity of pol delta. These results suggest that 3' to 5' exonuclease-dependent proofreading enhances the fidelity of DNA synthesis by a mammalian DNA polymerase in vitro.     

Okazaki fragment processing: Modulation of the strand displacement activity of DNA polymerase δ by the concerted action of replication protein A, proliferating cell nuclear antigen, and flap endonuclease-1

DNA polymerase (pol) delta is essential for both leading and lagging strand DNA synthesis during chromosomal replication in eukaryotes. Pol delta has been implicated in the Okazaki fragment maturation process for the extension of the newly synthesized fragment and for the displacement of the RNA/DNA segment of the preexisting downstream fragment generating an intermediate flap structure that is the target for the Dna2 and flap endonuclease-1 (Fen 1) endonucleases. Using a single-stranded minicircular template with an annealed RNA/DNA primer, we could measure strand displacement by pol delta coupled to DNA synthesis. Our results suggested that pol delta alone can displace up to 72 nucleotides while synthesizing through a double-stranded DNA region in a distributive manner. Proliferating cell nuclear antigen (PCNA) reduced the template dissociation rate of pol delta, thus increasing the processivity of both synthesis and strand displacement, whereas replication protein A (RP-A) limited the size of the displaced fragment down to 20-30 nucleotides, by generating a "locked" flap DNA structure, which was a substrate for processing of the displaced fragment by Fen 1 into a ligatable product. Our data support a model for Okazaki fragment processing where the strand displacement activity of DNA polymerase delta is modulated by the concerted action of PCNA, RP-A and Fen 1.     

Identification of a higher molecular weight DNA polymerase alpha catalytic polypeptide in monkey cells by monoclonal antibody.

A monoclonal antibody against purified calf DNA polymerase alpha (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) was used to immunoprecipitate proteins from a crude soluble extract of growing monkey BSC-1 cells. Immunoprecipitates contained familiar DNA polymerase alpha catalytic polypeptides of Mrs approximately equal to 115,000 and 70,000 and also a Mr 40,000 catalytic polypeptide; the major component in the immunoprecipitates, however, was a polypeptide of Mr approximately equal to 190,000 not previously identified as a DNA polymerase. This protein was capable of DNA polymerase activity after electroelution from NaDodSO4/polyacrylamide gels and renaturation. The highly purified enzyme so obtained was active with poly(dT).oligo(rA) as template.primer, resistant to dideoxy TTP (ddTTP), and inhibited by aphidicolin and butylphenyldeoxyguanosine 5'-triphosphate, thus identifying it as a DNA polymerase alpha. The results indicate that a polypeptide of Mr approximately equal to 190,000 is an abundant component among DNA polymerase alpha catalytic polypeptides in growing monkey cells.     

The delta subunit of Escherichia coli DNA polymerase III holoenzyme is the dnaX gene product.

The delta subunit of DNA polymerase III holoenzyme has been purified extensively with an assay for phi X174 DNA synthesis using core (pol III) and beta and gamma subunits. Either the purified delta subunit or the purified DNA polymerase III holoenzyme can complement a defective enzyme fraction from the conditional replication mutant SG133 described by Sevastopoulos et al. [Sevastopoulas, C.G., Wehr, C.T. & Glaser, D. A. (1977) Proc. Natl. Acad. Sci. USA 74, 3485-3489]. It has been established by Henson et al. [Henson, J.M., Chu, H., Irwin, C.A. & Walker, J.R. (1979) Genetics 92, 1,41-1059] that SG133 has two temperature-sensitive mutations, called dnaX and dnaY. The crude enzyme source from dnaX can be complemented by the delta subunit and by DNA polymerase III holoenzyme. By contrast, the core DNA polymerase III and the beta and gamma subunits are unable to complement this defective enzyme fraction. Thus, the delta subunit of DNA polymerase III holoenzyme appears to be the dnaX gene product of Escherichia coli.     

Ubiquitinated proliferating cell nuclear antigen activates translesion DNA polymerases eta and REV1

In response to DNA damage, the Rad6/Rad18 ubiquitin-conjugating complex monoubiquitinates the replication clamp proliferating cell nuclear antigen (PCNA) at Lys-164. Although ubiquitination of PCNA is recognized as an essential step in initiating postreplication repair, the mechanistic relevance of this modification has remained elusive. Here, we describe a robust in vitro system that ubiquitinates yeast PCNA specifically on Lys-164. Significantly, only those PCNA clamps that are appropriately loaded around effector DNA by its loader, replication factor C, are ubiquitinated. This observation suggests that, in vitro, only PCNA present at stalled replication forks is ubiquitinated. Ubiquitinated PCNA displays the same replicative functions as unmodified PCNA. These functions include loading onto DNA by replication factor C, as well as Okazaki fragment synthesis and maturation by the PCNA-coordinated actions of DNA polymerase delta, the flap endonuclease FEN1, and DNA ligase I. However, whereas the activity of DNA polymerase zeta remains unaffected by ubiquitination of PCNA, ubiquitinated PCNA specifically activates two key enzymes in translesion synthesis: DNA polymerase eta, the yeast Xeroderma pigmentosum ortholog, and Rev1, a deoxycytidyl transferase that functions in organizing the mutagenic DNA replication machinery. We propose that ubiquitination of PCNA increases its functionality as a sliding clamp to promote mutagenic DNA replication.