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.     

The subunits of activator 1 (replication factor C) carry out multiple functions essential for proliferating-cell nuclear antigen-dependent DNA synthesis.

p37 and p40 are two cloned gene products of the five-subunit human cellular DNA replication factor activator 1 (A1) protein complex (also called replication factor C). Here, we describe the solubilization, purification, and characterization of these two proteins that were overproduced in Escherichia coli. Using a nitrocellulose filter binding assay, we demonstrated that the purified A1 p37 protein associated with DNA preferentially at the primer terminus, a property resembling that of the A1 complex. We also show that in the presence of relatively high levels of salt, the recombinant p37 protein alone activated DNA polymerase epsilon but not polymerase delta in catalyzing the elongation of DNA chains. The p40 protein specifically associated with cellular p37 and proliferating-cell nuclear antigen (PCNA) present in HeLa cell cytosolic extract. The addition of purified p40 protein abolished the in vitro polymerase delta-catalyzed DNA elongation reaction dependent on both PCNA and A1. However, this inhibition was reversed by excess polymerase delta, suggesting a specific interaction between the polymerase and the p40 protein. Thus, while p37 binds DNA at the primer end and has a specific affinity for pol epsilon, p40, which binds ATP, interacts with PCNA and pol delta. These activities are essential for the DNA elongation reactions that lead to the synthesis of leading-strand DNA and the maturation of Okazaki fragments.     

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.     

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.     

Werner protein recruits DNA polymerase delta to the nucleolus

Werner syndrome is a Mendelian disorder of man that produces a number of manifestations resembling human aging. This disorder is caused by inactivation of the wrn gene, a member of the RecQ family of DNA helicases. The helicase and exonuclease activities of the Werner protein (WRN) suggest that it functions in DNA transactions, but the physiological function of WRN remains elusive. We present several lines of evidence that WRN interacts specifically with the p50 subunit of polymerase delta, the major DNA polymerase required for chromosomal DNA replication. P50, identified by yeast two-hybrid screening, interacts physically with the C terminus of WRN. Native WRN protein coimmunoprecipitates with p50 in a cellular fraction enriched in nucleolar proteins, and this immunocomplex also includes p125, the catalytic subunit of polymerase delta. In subcellular localization studies of cells transfected with WRN, p50 and p125 redistribute to the nucleolus and colocalize with WRN. These results suggest that one of the functions of WRN protein is to directly modify DNA replication via its interaction with p50 and abet dynamic relocalization of the DNA polymerase delta complexes within the nucleus.     

Requirement of proliferating cell nuclear antigen in RAD6-dependent postreplicational DNA repair.

The proliferating cell nuclear antigen (PCNA) acts as a processivity factor for replicative DNA polymerases and is essential for DNA replication. In vitro studies have suggested a role for PCNA-in the repair synthesis step of nucleotide excision repair, and PCNA interacts with the cyclin-dependent kinase inhibitor p21. However, because of the lack of genetic evidence, it is not clear which of the DNA repair processes are in fact affected by PCNA in vivo. Here, we describe a PCNA mutation, pol30-46, that confers ultraviolet (UV) sensitivity but has no effect on growth or cell cycle progression, and the mutant pcna interacts normally with DNA polymerase delta and epsilon. Genetic studies indicate that the pol30-46 mutation is specifically defective in RAD6-dependent postreplicational repair of UV damaged DNA, and this mutation impairs the error-free mode of bypass repair. These results implicate a role for PCNA as an intermediary between DNA replication and postreplicational DNA repair.     

An interaction between DNA ligase I and proliferating cell nuclear antigen: Implications for Okazaki fragment synthesis and joining

Although three human genes encoding DNA ligases have been isolated, the molecular mechanisms by which these gene products specifically participate in different DNA transactions are not well understood. In this study, fractionation of a HeLa nuclear extract by DNA ligase I affinity chromatography resulted in the specific retention of a replication protein, proliferating cell nuclear antigen (PCNA), by the affinity resin. Subsequent experiments demonstrated that DNA ligase I and PCNA interact directly via the amino-terminal 118 aa of DNA ligase I, the same region of DNA ligase I that is required for localization of this enzyme at replication foci during S phase. PCNA, which forms a sliding clamp around duplex DNA, interacts with DNA pol δ and enables this enzyme to synthesize DNA processively. An interaction between DNA ligase I and PCNA that is topologically linked to DNA was detected. However, DNA ligase I inhibited PCNA-dependent DNA synthesis by DNA pol δ. These observations suggest that a ternary complex of DNA ligase I, PCNA and DNA pol δ does not form on a gapped DNA template. Consistent with this idea, the cell cycle inhibitor p21, which also interacts with PCNA and inhibits processive DNA synthesis by DNA pol δ, disrupts the DNA ligase I–PCNA complex. Thus, we propose that after Okazaki fragment DNA synthesis is completed by a PCNA–DNA pol δ complex, DNA pol δ is released, allowing DNA ligase I to bind to PCNA at the nick between adjacent Okazaki fragments and catalyze phosphodiester bond formation.     

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.     

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.     

Suppression of cell transformation by the cyclin-dependent kinase inhibitor p57KIP2 requires binding to proliferating cell nuclear antigen

Proper control of the mammalian cell cycle requires the function of cyclin-dependent kinase (CDK) inhibitors. The p21 family currently includes three distinct genes, p21, p27^Kip1, and p57^Kip2, that share a common N-terminal domain for binding to and inhibiting the kinase activity of CDK-cyclin complexes. The p21 protein also binds to proliferating cell nuclear antigen (PCNA) through a separate C-terminal domain affecting DNA replication and repair. The p27 and p57 proteins also each contain unique C-terminal domains whose functions are unknown. Here we show that the human p57 protein, like p21, contains a PCNA-binding domain within its C terminus that, when separated from its N-terminal CDK-cyclin binding domain, can prevent DNA replication in vitro and S phase entry in vivo. Disruption of either CDK/cyclin or PCNA binding partially reduced p57’s ability to suppress myc/RAS-mediated transformation in primary cells, while loss of both inhibitory functions completely eliminated p57’s suppressive activity. Thus, control of cell cycle and suppression of cell transformation by p57 require both CDK and PCNA inhibitory activity, and disruption of either or both functions may lead to uncontrolled cell growth.     

Interaction of the β sliding clamp with MutS, ligase, and DNA polymerase I

The beta and proliferating cell nuclear antigen (PCNA) sliding clamps were first identified as components of their respective replicases, and thus were assigned a role in chromosome replication. Further studies have shown that the eukaryotic clamp, PCNA, interacts with several other proteins that are involved in excision repair, mismatch repair, cellular regulation, and DNA processing, indicating a much wider role than replication alone. Indeed, the Escherichia coli beta clamp is known to function with DNA polymerases II and V, indicating that beta also interacts with more than just the chromosomal replicase, DNA polymerase III. This report demonstrates three previously undetected protein-protein interactions with the beta clamp. Thus, beta interacts with MutS, DNA ligase, and DNA polymerase I. Given the diverse use of these proteins in repair and other DNA transactions, this expanded list of beta interactive proteins suggests that the prokaryotic beta ring participates in a wide variety of reactions beyond its role in chromosomal replication.     

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.     

Initiation of enzymatic replication at the origin of the Escherichia coli chromosome: contributions of RNA polymerase and primase.

Replication of plasmids that depend on the 245-base-pair origin of the Escherichia coli chromosome (oriC) requires many purified proteins that (i) direct initiation to oriC (e.g., dnaA protein), (ii) influence initiations elsewhere (e.g., auxiliary proteins), and (iii) prime and extend DNA chains (e.g., priming and synthesis proteins). For the RNA priming and initiation of new DNA chains, the requirements for both primase and RNA polymerase (RNA pol) [Kaguni, J. M. & Kornberg, A. (1984) Cell 38, 183-190] have been further analyzed. Depending on the levels of auxiliary proteins (topoisomerase I and protein HU), three priming systems can operate: primase alone, RNA pol alone, or both combined. At low levels of auxiliary proteins, primase alone sustains an effective priming system. At higher levels, primase action is blocked, but RNA pol alone can initiate replication, albeit feebly; at these high levels of auxiliary proteins, primase and RNA pol act synergistically. When RNA pol is stalled by an inhibitor or lack of a ribonucleoside triphosphate, primase action is also inhibited. Based on these and other data [van der Ende, A., Baker, T. A., Ogawa, T. & Kornberg, A. (1985) Proc. Natl. Acad. Sci. USA 82, in press], RNA pol can counteract inhibition by auxiliary proteins and thus activate the origin for the priming by primase of the leading strand of the replication fork.     

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.     

PCNA in the testis of the frog, Rana esculenta: a molecular marker of the mitotic testicular epithelium proliferation

Proliferating cell nuclear antigen (PCNA) plays an essential role in nucleic acid metabolism as a component of the replication and repair machinery. This toroidal-shaped protein encircles DNA and can slide bidirectionally along the duplex. One of the well-established functions for PCNA is its role as the processing factor for DNA polymerase delta and epsilon. It has become apparent that PCNA interacts with proteins involved in the cell cycle. The PCNA interactions with different cellular proteins and the importance of these interactions are also discussed. To examine the different mitotic testicular epithelium proliferation during the annual discontinuous frog (Rana esculenta) spermatogenesis, the temporal and the spatial PCNA expression are described and give a useful endogenous molecular marker.     

The fifth essential DNA polymerase phi in Saccharomyces cerevisiae is localized to the nucleolus and plays an important role in synthesis of rRNA

We report that POL5 encodes the fifth essential DNA polymerase in Saccharomyces cerevisiae. Pol5p was identified and purified from yeast cell extracts and is an aphidicolin-sensitive DNA polymerase that is stimulated by yeast proliferating cell nuclear antigen (PCNA). Thus, we named Pol5p DNA polymerase phi. Temperature-sensitive pol5-1-- -3 mutants did not arrest at G(2)/M at the restrictive temperature. Furthermore, the polymerase active-site mutant POL5dn gene complements the lethality of Delta pol5. These results suggest that the polymerase activity of Pol5p is not required for the in vivo function of Pol5p. rRNA synthesis was severely inhibited at the restrictive temperature in the temperature-sensitive pol5-3 mutant cells, suggesting that an essential function of Pol5p is rRNA synthesis. Pol5p is localized exclusively to the nucleolus and binds near or at the enhancer region of rRNA-encoding DNA repeating units.     

An inhibitor of the in vitro elongation reaction of simian virus 40 DNA replication is overcome by proliferating-cell nuclear antigen.

The replication of simian virus 40 (SV40) origin-containing DNA has been reconstituted by using SV40 large tumor (T) antigen and cellular proteins purified from HeLa cells. This replication reaction is unaffected by proliferating-cell nuclear antigen (PCNA). In contrast, PCNA has been reported to stimulate SV40 DNA synthesis carried out with crude fractions [Prelich, G., Kostura, M., Marshak, D. R., Mathews, M. B. & Stillman, B. (1987) Nature (London) 326, 471-475]. This difference is caused by the presence of a protein in crude fractions that inhibits the elongation of nascent DNA chains during replication. In the presence of PCNA, crude fractions containing this elongation inhibition factor can extend DNA chains. We describe the partial purification of this inhibitor and show that its addition limited SV40 DNA replication to the synthesis of short chains, an effect reversed by the addition of PCNA. However, the reversal of the inhibition by PCNA in the SV40 system required additional protein fractions distinct from PCNA and the enzymes constituting the purified system. These results suggest that the PCNA-mediated effect on SV40 DNA replication may be indirect. Such an interplay between negative and positive regulatory functions including PCNA may contribute to the control of DNA synthesis characteristic of the eukaryotic cell cycle.