Studies on the initiation and elongation reactions in the simian virus 40 DNA replication system.

The synthesis of oligoribonucleotides by DNA primase in the presence of duplex DNA containing the simian virus 40 (SV40) origin of replication was examined. Small RNA chains (10-15 nucleotides) were synthesized in the presence of the four common ribonucleoside triphosphates, SV40 large tumor antigen (T antigen), the human DNA polymerase alpha (pol alpha)-DNA primase complex, the human single-stranded DNA-binding protein (HSSB), and topoisomerase I isolated from HeLa cells. The DNA primase-catalyzed reaction showed an absolute requirement for T antigen, HSSB, and pol alpha. The requirement for HSSB was not satisfied by other SSBs that can support the T-antigen-catalyzed unwinding of DNA containing the SV40 origin of replication. Oligoribonucleotide synthesis occurred with a lag that paralleled the lag observed in DNA synthesis. These results indicate that the specificity for the HSSB in the SV40 replication reaction is due to the pol alpha-primase-mediated synthesis of the Okazaki fragments. In contrast to this specificity, the elongation of Okazaki fragments can be catalyzed by a variety of different DNA polymerases, including high levels of pol alpha, the polymerase delta holoenzyme, T4 polymerase holoenzyme, the Escherichia coli polymerase III holoenzyme, and other polymerases. These observations suggest that leading-strand synthesis in the in vitro SV40 replication system can be nonspecific.     

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.     

Conservation of structure and function of DNA replication protein A in the trypanosomatid Crithidia fasciculata.

Human replication protein A (RP-A) is a three-subunit protein that is required for simian virus 40 (SV40) replication in vitro. The trypanosome homologue of RP-A has been purified from Crithidia fasciculata. It is a 1:1:1 complex of three polypeptides of 51, 28, and 14 kDa, binds single-stranded DNA via the large subunit, and is localized within the nucleus. C. fasciculata RP-A substitutes for human RP-A in the large tumor antigen-dependent unwinding of the SV40 origin of replication and stimulates both DNA synthesis and DNA priming by human DNA polymerase alpha/primase, but it does not support efficient SV40 DNA replication in vitro. This extraordinary conservation of structure and function between human and trypanosome RP-A suggests that the mechanism of DNA replication, at both the initiation and the elongation level, is conserved in organisms that diverged from the main eukaryotic lineage very early in evolution.     

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.     

Reconstitution of human replication factor C from its five subunits in baculovirus-infected insect cells

Human replication factor C (RFC, also called activator 1) is a five-subunit protein complex (p140, p40, p38, p37, and p36) required for proliferating cell nuclear antigen (PCNA)-dependent processive DNA synthesis catalyzed by DNA polymerase δ or . Here we report the reconstitution of the RFC complex from its five subunits simultaneously overexpressed in baculovirus-infected insect cells. The purified baculovirus-produced RFC appears to contain equimolar levels of each subunit and was shown to be functionally identical to its native counterpart in (i) supporting DNA polymerase δ-catalyzed PCNA-dependent DNA chain elongation; (ii) catalyzing DNA-dependent ATP hydrolysis that was stimulated by PCNA and human single-stranded DNA binding protein; (iii) binding preferentially to DNA primer ends; and (iv) catalytically loading PCNA onto singly nicked circular DNA and catalytically removing PCNA from these DNA molecules.     

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.     

Multiple functions of human single-stranded-DNA binding protein in simian virus 40 DNA replication: single-strand stabilization and stimulation of DNA polymerases alpha and delta.

The human single-stranded-DNA binding protein (human SSB) is required for simian virus 40 (SV40) DNA replication in vitro. SV40 large tumor antigen and human SSB can support extensive unwinding of SV40 origin-containing DNA in the presence of ATP and a topoisomerase that relieves positive superhelicity. Although SSBs from viral and prokaryotic sources substituted for human SSB in the DNA-unwinding reaction, they did not substitute in the replication of SV40 DNA. The specificity for human SSB in SV40 DNA replication can be explained, at least in part, by the finding that DNA polymerase alpha was stimulated 10-fold by human SSB but not by other SSBs. Human SSB also stimulated proliferating-cell nuclear antigen-dependent DNA polymerase delta; however, other SSBs stimulated this polymerase as well.     

Reconstitution of simian virus 40 DNA replication with purified proteins.

Replication of plasmid DNA molecules containing the simian virus 40 (SV40) origin of DNA replication has been reconstituted with seven highly purified cellular proteins plus the SV40 large tumor (T) antigen. Initiation of DNA synthesis is absolutely dependent upon T antigen, replication protein A, and the DNA polymerase alpha-primase complex and is stimulated by the catalytic subunit of protein phosphatase 2A. Efficient elongation of nascent chains additionally requires proliferating cell nuclear antigen, replication factor C, DNA topoisomerase I, and DNA polymerase delta. Electron microscopic studies indicate that DNA replication begins at the viral origin and proceeds via intermediates containing two forks that move in opposite directions. These findings indicate that the reconstituted replication reaction has many of the characteristics expected of authentic viral DNA replication.     

Functions of replication factor C and proliferating-cell nuclear antigen: functional similarity of DNA polymerase accessory proteins from human cells and bacteriophage T4.

The proliferating-cell nuclear antigen (PCNA) and the replication factors A and C (RF-A and RF-C) are cellular proteins essential for complete elongation of DNA during synthesis from the simian virus 40 origin of DNA replication in vitro. All three cooperate to stimulate processive DNA synthesis by DNA polymerase delta on a primed single-stranded M13 template DNA and as such can be categorized as DNA polymerase accessory proteins. Biochemical analyses with highly purified RF-C and PCNA have demonstrated functions that are completely analogous to the functions of bacteriophage T4 DNA polymerase accessory proteins. A primer-template-specific DNA binding activity and a DNA-dependent ATPase activity copurified with the multisubunit protein RF-C and are similar to the functions of the phage T4 gene 44/62 protein complex. Furthermore, PCNA stimulated the RF-C ATPase activity and is, therefore, analogous to the phage T4 gene 45 protein, which stimulates the ATPase function of the gene 44/62 protein complex. Indeed, some primary sequence similarities between human PCNA and the phage T4 gene 45 protein could be detected. These results demonstrate a striking conservation of the DNA replication apparatus in human cells and bacteriophage T4.     

Replication of simian virus 40 origin-containing DNA in vitro with purified proteins.

Simian virus 40 (SV40) DNA replication dependent on the SV40 origin of replication and the SV40 large tumor (T) antigen has been reconstituted in vitro with purified protein components isolated from HeLa cells. In addition to SV40 T antigen, these components included the DNA polymerase alpha-primase complex, topoisomerase I, and a fraction that contained a single-stranded DNA binding protein. The latter protein, which sediments at 5.1 S on glycerol gradients and copurifies with two major protein species of 72 and 76 kDa, was isolated solely by its ability to support SV40 DNA replication. The purified system retained the species-specific DNA polymerase alpha-primase requirement previously observed with crude fractions; the complex from HeLa cells supported SV40 replication, whereas that from calf thymus and mouse cells did not. DNA containing the polyomavirus origin of replication was replicated in a system containing polyomavirus T antigen, the HeLa single-stranded DNA binding protein-containing fraction, and DNA polymerase alpha-primase complex from mouse, but not HeLa, cells. While crude fractions yielded closed circular duplex DNA, none was detected with the purified system. Nevertheless, the addition of a crude fraction to the purified system yielded closed circular monomer products.     

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.     

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.     

Purification and characterization of replication protein A, a cellular protein required for in vitro replication of simian virus 40 DNA.

The replication of simian virus 40 (SV40) DNA is largely dependent upon cellular replication proteins. To define these proteins we have made use of a cell-free system that is capable of replicating plasmid DNA molecules containing the SV40 origin of replication. Systematic fractionation-reconstitution experiments indicate that there are a minimum of six cellular proteins that are required for efficient viral DNA replication in vitro. We report here the purification of one of these proteins, replication protein A (RP-A), to homogeneity. RP-A is a multisubunit protein that contains four tightly associated polypeptides of 70, 53, 32, and 14 kDa. Partial proteolysis experiments indicate that the 53-kDa polypeptide is closely related to the 70-kDa polypeptide, suggesting that it may be a proteolytic fragment of the larger subunit. RP-A is absolutely required for reconstitution of SV40 DNA replication in vitro. The purified protein binds to single-stranded DNA and is required for the large tumor (T)-antigen-mediated unwinding of DNA molecules containing the SV40 origin of DNA replication. These properties are consistent with the possibility that RP-A plays a central role in the generation of a single-stranded region at the origin prior to initiation of DNA synthesis. The protein may also function to facilitate unwinding of the parental DNA strands during the elongation phase of SV40 DNA replication.     

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.     

Initiation of simian virus 40 DNA replication in vitro: large-tumor-antigen- and origin-dependent unwinding of the template.

Analysis of the kinetics of simian virus 40 (SV40) DNA replication in vitro demonstrated the existence of a slow presynthesis reaction that occurs prior to onset of extensive chain elongation and is dependent on a subset of the cellular proteins required for the complete replication reaction. When the presynthesis reaction is carried out in the presence of topoisomerase I, it is possible to detect extensive unwinding of the template DNA. This unwinding reaction is specific for templates that contain the wild-type SV40 origin of DNA replication and requires SV40 large tumor antigen (T antigen), ATP, and a protein fraction derived from HeLa cells. The required cellular protein may be a eukaryotic single-stranded-DNA-binding protein (SSB), since unwinding of the template is also observed when Escherichia coli SSB is substituted for the HeLa protein fraction. These observations suggest that during the initial stages of SV40 DNA replication, T antigen binds specifically to the viral origin and locally unwinds the DNA. This origin-dependent unwinding reaction is presumably a prerequisite for subsequent priming and elongation steps.     

Requirement for two DNA polymerases in the replication of simian virus 40 DNA in vitro.

DNA polymerase alpha-primase has long been considered the primary, if not sole, replicative DNA polymerase in eukaryotic cells. However, recent experiments have provided indirect evidence that a second DNA polymerase may play a role in DNA replication. To identify cellular proteins necessary for DNA synthesis in mammalian cells, we have been studying the cell-free system developed for the replication of simian virus 40 DNA. In this report, we present direct evidence that a second DNA polymerase is required in addition to DNA polymerase alpha-primase complex to obtain efficient replication of simian virus 40 origin-containing DNA. This DNA polymerase activity is not affected by monoclonal antibodies that inhibit the activity of DNA polymerase alpha and is relatively resistant to the inhibitor [N2-(p-n-butylphenyl)-9-(2-deoxy-beta-D-ribofuranosyl)guanine 5'-triphosphate]. Moreover, the activity of the polymerase is highly dependent upon the accessory protein, proliferating-cell nuclear antigen. These characteristics are consistent with the hypothesis that this second DNA polymerase is DNA polymerase delta.     

Involvement of escherichia coli dnaZ gene product in DNA elongation in vitro.

E. coli dnaZ gene product is required for conversion of phiX174, fd, and ST-1 single-stranded phage DNAs to duplex DNAs in vitro. This protein has been purified about 5000-fold. It functions in the elongation of RNA- or DNA-primed single-stranded DNA that is catalyzed by DNA polymerase III(DNA nucleotidyltransferase; deoxynucleosidetriphosphate: DNA deoxynucleotidyltransferase; EC 2.7.7.7) in conjunctions with two other E. coli protein preparations referred to as DNA elongation factors I and III. It also functions in similar reactions catalyzed by DNA polymerase II in combination with E. coli DNA binding protein and DNA elongation factors I and III.     

Model system for DNA replication of a plasmid DNA containing the autonomously replicating sequence from Saccharomyces cerevisiae.

A negatively supercoiled plasmid DNA containing autonomously replicating sequence (ARS) 1 from Saccharomyces cerevisiae was replicated with the proteins required for simian virus 40 DNA replication. The proteins included simian virus 40 large tumor antigen as a DNA helicase, DNA polymerase alpha.primase, and the multisubunit human single-stranded DNA-binding protein from HeLa cells; DNA gyrase from Escherichia coli, which relaxes positive but not negative supercoils, was included as a "swivelase." DNA replication started from the ARS region, proceeded bidirectionally with the synthesis of leading and lagging strands, and resulted in the synthesis of up to 10% of the input DNA in 1 h. The addition of HeLa DNA topoisomerase I, which relaxes both positive and negative supercoils, to this system inhibited DNA replication, suggesting that negative supercoiling of the template DNA is required for initiation. These results suggest that DNA replication starts from the ARS region where the DNA duplex is unwound by torsional stress; this unwound region can be recognized by a DNA helicase with the assistance of the multisubunit human single-stranded DNA-binding protein.     

Adenovirus DNA replication in vitro: synthesis of full-length DNA with purified proteins.

A protein required for the elongation of replicating intermediates of adenovirus (Ad) DNA to full length has been isolated and characterized. This factor, isolated from nuclear extracts of uninfected HeLa cells, has been designated nuclear factor II. In the presence of Ad DNA with proteins at each 5' end (Ad DNA-protein) and three proteins coded for by the Ad genome [the preterminal protein (pTP), the DNA polymerase (Ad Pol), and the DNA binding protein (Ad DBP)], nuclear factor II complementing activity is detected only in the presence of host nuclear factor I. Highly purified preparations of nuclear factor II that are free of detectable DNA polymerase alpha, beta, and gamma activities contain a DNA topoisomerase activity. Furthermore, type I DNA topoisomerases purified from HeLa cells and calf thymus substitute for nuclear factor II complementing activity in the in vitro Ad DNA replication system. These results indicate that a protein that is involved in higher order DNA structure is required for Ad replication. This protein plus the purified proteins described above carry out the initiation and synthesis of full-length 36,000-base-pair Ad DNA.