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At least 379 records · Page 21Linked to original sources
PubMed2006

DNA polymerase-beta is expressed early in neurons of Alzheimer's disease brain and is loaded into DNA replication forks in neurons challenged with beta-amyloid.

Cultured neurons exposed to synthetic beta-amyloid (Abeta) fragments reenter the cell cycle and initiate a pathway of DNA replication that involves the repair enzyme DNA polymerase-beta (DNA pol-beta) before undergoing apoptotic death. In this study, by performing coimmunoprecipitation experiments on cross-linked nucleoprotein fragments from Abeta-treated neurons, we demonstrate that DNA pol-beta coimmunoprecipitates with cell division cycle 45 (Cdc45) and with DNA primase in short nucleoprotein fragments. This indicates that DNA pol-beta is loaded into neuronal DNA replication forks after Abeta treatment. In response to Abeta the canonical DNA-synthesizing enzyme DNA pol-delta also was loaded into neuronal replication forks, but at later times than DNA pol-beta. Methoxyamine, an inhibitor of the apurinic/apyrimidinic endonuclease that allows for the recruitment of DNA pol-beta during the process of base excision repair (BER), failed to affect coimmunoprecipitation between DNA pol-beta and Cdc45, indicating that DNA pol-beta loading to the replication forks is independent of DNA breaks. However, methoxyamine reduced DNA replication and ensuing apoptosis in neurons exposed to Abeta, suggesting that an efficient BER process allows DNA replication to proceed up to the threshold for death. These data demonstrate that DNA pol-beta is an essential component of the DNA replication machinery in Abeta-treated neurons and additionally support the hypothesis of a close association of cell cycle events with neuronal death in Alzheimer's disease (AD). Accordingly, by investigating the neuronal expression of DNA pol-beta, along with phosphorylated retinoblastoma protein and neurofibrillary changes in AD brain, we show an early involvement of DNA pol-beta in the pathogenesis of AD.

Adult↗
PubMed1988

DNA polymerase alpha-primase from calf thymus. Determination of the polypeptide responsible for primase activity.

Immunoaffinity-purified DNA polymerase alpha-primase complex from calf thymus consists of subunits with molecular weights of 148,000-180,000, 73,000, 59,000, and 48,000 (Nasheuer, H.-P., and Grosse, F. (1987) Biochemistry 26, 8458-8466). Primase activity was separated from the immobilized complex by washing extensively with 2 M KCl or, alternatively, by shifting to pH 11.5 in the presence of 1 M KCl. From both elution procedures, the primase activity was found to be associated with the polypeptides with molecular weights of 59,000 and 48,000. The specific activity, using either elution procedure, was 30,000 units/mg. Both polypeptides sedimented together at 5.7 S upon zonal centrifugation on a sucrose gradient. Primase activity was found in the flow-through fraction after DEAE-cellulose chromatography of the free primase. Analysis of this fraction by sodium dodecyl sulfate gel electrophoresis revealed only one band with a Mr of 48,000. Polyclonal antibodies were raised against the Mr 59,000 and 48,000 polypeptides. The anti-Mr 59,000 antibody affected the primase activity only marginally, whereas the anti-Mr 48,000 antibody inhibited the primase activity nearly completely. UV cross-linking of the DNA polymerase alpha-primase complex with alpha-32P-labeled GTP revealed a binding site at the Mr 48,000 polypeptide, but none at the other subunits of the complex. Taken together, these results suggest that the Mr 48,000 polypeptide bears the active site of the DNA primase activity. The Mr 59,000 polypeptide stabilizes the primase activity.

Animals↗
PubMed2001

Role of fission yeast primase catalytic subunit in the replication checkpoint.

To investigate the cell cycle checkpoint response to aberrant S phase-initiation, we analyzed mutations of the two DNA primase subunit genes of Schizosaccharomyces pombe, spp1(+) and spp2(+) (S. pombe primase 1 and 2). spp1(+) encodes the catalytic subunit that synthesizes the RNA primer, which is then utilized by Polalpha to synthesize the initiation DNA. Here, we reported the isolation of the fission yeast spp1(+) gene and cDNA and the characterization of Spp1 protein and its cellular localization during the cell cycle. Spp1 is essential for cell viability, and thermosensitive mutants of spp1(+) exhibit an allele-specific abnormal mitotic phenotype. Mutations of spp1(+) reduce the steady-state cellular levels of Spp1 protein and compromised the formation of Polalpha-primase complex. The spp1 mutant displaying an aberrant mitotic phenotype also fails to properly activate the Chk1 checkpoint kinase, but not the Cds1 checkpoint kinase. Mutational analysis of Polalpha has previously shown that activation of the replication checkpoint requires the initiation of DNA synthesis by Polalpha. Together, these have led us to propose that suboptimal cellular levels of polalpha-primase complex due to the allele-specific mutations of Spp1 might not allow Polalpha to synthesize initiation DNA efficiently, resulting in failure to activate a checkpoint response. Thus, a functional Spp1 is required for the Chk1-mediated, but not the Cds1-mediated, checkpoint response after an aberrant initiation of DNA synthesis.

Amino Acid Sequence↗
PubMed1995

Further characterization of the human cell multiprotein DNA replication complex.

Evidence for multiprotein complexes playing a role in DNA replication has been growing over the years. We have previously reported on a replication-competent multiprotein form of DNA polymerase isolated from human (HeLa) cell extracts. The proteins that were found at that time to co-purify with the human cell multiprotein form of DNA polymerase included: DNA polymerase alpha, DNA primase, topoisomerase I, RNase H, PCNA, and a DNA-dependent ATPase. The multiprotein form of the human cell DNA polymerase was further purified by Q-Sepharose chromatography followed by glycerol gradient sedimentation and was shown to be fully competent to support origin-specific and large T-antigen dependent simian virus 40 (SV40) DNA replication in vitro [Malkas et al. (1990b): Biochemistry 29:6362-6374]. In this report we describe the further characterization of the human cell replication-competent multiprotein form of DNA polymerase designated MRC. Several additional DNA replication proteins that co-purify with the MRC have been identified. These proteins include: DNA polymerase delta, RF-C, topoisomerase II, DNA ligase I, DNA helicase, and RP-A. The replication requirements, replication initiation kinetics, and the ability of the MRC to utilize minichromosome structures for DNA synthesis have been determined. We also report on the results of experiments to determine whether nucleotide metabolism enzymes co-purify with the human cell MRC. We recently proposed a model to represent the MRC that was isolated from murine cells [Wu et al. (1994): J Cell Biochem 54:32-46]. We can now extend this model to include the human cell MRC based on the fractionation, chromatographic and sedimentation behavior of the human cell DNA replication proteins. A full description of the model is discussed. Our experimental results provide further evidence to suggest that DNA synthesis is mediated by a multiprotein complex in mammalian cells.

Blotting, Western↗
PubMed1984

Sequence specificity for the initiation of RNA-primed simian virus 40 DNA synthesis in vivo.

Analysis of the nucleotide sequences at the 5' ends of RNA-primed nascent DNA chains (Okazaki fragments) and of their locations in replicating simian virus 40 (SV40) DNA revealed the precise nature of Okazaki fragment initiation sites in vivo. The primary initiation site for mammalian DNA primase was 3'-purine-dT-5' in the DNA template and the secondary site was 3'-purine-dC-5', with the 5' end of the RNA primer complementary to either the dT or dC. The third position of the initiation site was variable with a preference for dT or dA. About 81% of the available 3'-purine-dT-5' sites and 20% of the 3'-purine-dC-5' sites were used. Purine-rich sites, such as PuPuPu and PyPuPu , were excluded. The 5'-terminal ribonucleotide composition of Okazaki fragments corroborated these conclusions. Furthermore, the length of individual RNA primers was not unique, but varied in size from six to ten bases with some appearing as short as three bases and some as long as 12 bases, depending on the initiation site used. This result was consistent with the average size (9 to 11 bases) of RNA primers isolated from specific regions of the genome. Excision of RNA primers did not appear to stop at the RNA-DNA junction, but removed a variable number of deoxyribonucleotides from the 5' end of the nascent DNA chain. Finally, only one-fourth of the replication forks contained an Okazaki fragment, and the distribution of their initiation sites between the two arms revealed that Okazaki fragments were initiated exclusively (99%) on retrograde DNA templates. The data obtained at two genomic sites about 350 and 1780 bases from ori were essentially the same as that reported for the ori region (Hay & DePamphilis , 1982), suggesting that the mechanism used to synthesize the first DNA chain at ori is the same as that used to synthesize Okazaki fragments throughout the genome.

Base Sequence↗
PubMed2004

The utility of a two-color fluorescence electrophoretic mobility shift assay procedure for the analysis of DNA replication complexes.

Protein-DNA and protein-protein interactions play essential roles in many biologic processes such as transcription, replication, recombination, and DNA repair. One of the most popular approaches to investigate specific protein-nucleic acid interactions is the electrophoretic mobility shift assay (EMSA). We have developed a new nonradioactive method to detect both nucleotides and protein in EMSA. Nucleic acids are detected with SYBR Green EMSA dye, while proteins are subsequently detected with SYPRO Ruby EMSA dye. All fluorescent staining steps are performed after the entire gel-shift experiment is completed, so there is no need to prelabel either the nucleic acids or the protein. The two-color fluorescence dye staining procedure is fast, simple, and allows independent quantitative determination of either free or bound nucleic acids and protein. The interactions between lac repressor-operator, and among the T4 primase-helicase, primase-DNA, helicase-DNA, and within T4 [ssDNA-primase-helicase6] primosome, were used to demonstrate the advantages of this two-color fluorescence detection EMSA method.

Bacteriophage T4↗
PubMed1992

Cloning of the gene for the 73 kD subunit of the DNA polymerase alpha primase of Drosophila melanogaster.

We have isolated both cDNA and genomic clones for the 73 kDa subunit of the DNA polymerase alpha primase of Drosophila melanogaster. Analysis of these clones has identified an open reading frame of 1959 bases coding for a protein of 72.5 kDa. Northern analysis has shown the mRNA for the gene to be approximately 2.5 kb, and comparison of the cDNA and the genomic clones shows that the coding region of the gene lacks introns. The 5' end of the transcript has been mapped by primer extension, and the position of the gene in the genome mapped using in situ analysis. Computer analysis has been carried out on both coding and non coding regions of the gene. The protein sequence shows some homology to the analogous subunit in the S. cerevisiae DNA polymerase alpha, however a search of the data banks failed to reveal other homologies, or provide any clues as to the function of the protein. Analysis of the non-coding regions indicates some potential control regions for the gene. The 73 kDa protein has been overproduced, but a preliminary analysis failed to reveal any enzymatic activities.

Amino Acid Sequence↗
PubMed1991

DNA polymerase B from wheat embryos: a plant delta-like DNA polymerase.

Studies in eucaryotic cells (mainly animals and yeast) indicate that at least two DNA polymerases are involved in DNA replication at the level of the replication fork: DNA polymerase alpha, which is associated with DNA primase, is involved in the replication of the lagging strand; DNA polymerase delta, associated with an exonuclease activity, synthesizes the forward continuous DNA strand. Much less information exists concerning plant systems. Previous work from this laboratory provided preliminary evidence of an association between DNA polymerase B from wheat embryo and an exonucleolytic activity. In this paper, we present additional data on the biochemical properties of DNA polymerase B. An improved purification procedure described in this article has been developed. During all the purification steps the nuclease activity was associated with DNA polymerase activity. A biochemical study of this enzyme activity shows that it is an exonuclease which hydrolyses DNA in the 3' to 5' direction. Moreover, this exonuclease confers a proofreading function to DNA polymerase B. Comparison of DNA polymerase B properties (template specificity, sensitivity to DNA replication inhibitors like aphidicolin and butyl-phenyl dGTP, copurification of DNA polymerase and exonuclease activities) with those of animal DNA polymerase delta indicates that these enzymes share many common features. To our knowledge, this is the first report of DNA polymerase delta in higher plants.

Adenosine Monophosphate↗
PubMed1992

In vitro cleavage of double- and single-stranded DNA by plasmid RSF1010-encoded mobilization proteins.

We have used purified RSF1010 mobilization proteins to reproduce in vitro a strand-specific nicking at the plasmid origin of transfer, oriT. In the presence of Mg2+, the proteins MobA (78-kDa form of RSF1010 DNA primase), MobB, and MobC and supercoiled or linear duplex oriT DNA form large amounts of a cleavage complex, which is characterized by its sensitivity to protein-denaturant treatment. Upon addition of SDS to such a complex, a single strand break is generated in the DNA, and MobA is found linked to the 5' nick terminus, presumably covalently. The double-strand nicking activity of MobA requires, in addition to Mg2+, the presence of MobC and is stimulated by the presence of MobB. The nick site has been shown by DNA sequencing to lie at the position cleaved in vivo during transfer, between nucleotides 3138/3139 in the r strand of RSF1010. We have found that MobA will also cleave DNA at sites other than oriT if the DNA is present in single-stranded form. Breakage in this case occurs in the absence of denaturing conditions, and after prolonged incubation, reclosure can be demonstrated.

Bacterial Proteins↗
PubMed2003

DNA-protein interactions during the initiation and termination of plasmid pT181 rolling-circle replication.

Initiation of DNA replication requires the generation of a primer at the origin of replication that can be utilized by a DNA polymerase for DNA synthesis. This can be accomplished by several means, including the synthesis of an RNA primer by a DNA primase or RNA polymerase, by nicking of one strand of the DNA to generate a free 3'-OH end that can be used as a primer, and by the utilization of the OH group present in an amino acid such as serine within an initiation protein as a primer. Furthermore, some single-stranded DNA genomes can utilize a snap-back 3'-OH end generated due to self-complementarity as a primer for DNA replication. The different modes of initiation require the generation of highly organized DNA-protein complexes at the origin that trigger the initiation of replication. A large majority of small, multicopy plasmids of Gram-positive bacteria and some of Gram-negative bacteria replicate by a rolling-circle (RC) mechanism (for previous reviews, see Refs.). More than 200 rolling-circle replicating (RCR) plasmids have so far been identified and, based on sequence homologies in their replication regions, can be grouped into approximately seven families (Refs., and http://www.essex.ac.uk/bs/staff/osborn/DPR-home.htm). This review will focus on plasmids of the pT181 family that replicate by an RC mechanism. So far, approximately 25 plasmids have been identified as belonging to this family based on the sequence homology in their double-strand origins (dsos) and the genes encoding the initiator (Rep) proteins. This review will highlight our current understanding of the structural features of the origins of replication, and the DNA-protein and protein-protein interactions that result in the generation of a replication-initiation complex that triggers replication. It will discuss the molecular events that result in the precise termination of replication once the leading-strand DNA synthesis has been completed. This review will also discuss the various biochemical activities of the initiator proteins encoded by the plasmids of the pT181 family and the mechanism of inactivation of the Rep activity after supporting one round of leading-strand replication. Finally, the review will outline the mechanism of replication of the lagging strand of the pT181 plasmid as well as the limited information that is available on the role of host proteins in pT181 leading- and lagging-strand replication.

Amino Acid Sequence↗
PubMed1985

The primase activity of DNA polymerase alpha from calf thymus.

The nearly homogeneous 9 S DNA polymerase alpha from calf thymus contains a primase activity that allows priming of DNA synthesis on single-stranded templates in the presence of ribonucleoside triphosphates. Both on synthetic and natural single-stranded templates, RNA primers of 8-15 nucleotides in length are formed. In the absence of dNTPs, primers of some hundred nucleotides in length are observable. ATP and/or GTP are required for the priming reaction. UTP and CTP cannot initiate the RNA synthesis. M13 single-stranded DNA can be converted to the nicked double helical form upon primase-primed replication by the 9 S enzyme. Priming occurs mostly at specific sites on the M13 genome and replication products of up to 6000 nucleotides in length are formed. In the presence of the single-stranded DNA binding protein from Escherichia coli, specificity of priming is strongly increased. The primase is inhibited by salt and actinomycin; it is insensitive to alpha-amanitin and N-ethylmaleimide. Due to the strong complex formation between DNA polymerase and primase, it has not been possible to separate the two activities of the multisubunit 9 S enzyme.

Adenosine Triphosphate↗
PubMed1985

Dissociation and reconstitution of a DNA polymerase alpha-primase complex.

The conditions for dissociation of the DNA polymerase alpha-primase complex (DNA polymerase alpha 1) have been examined. It was revealed that 50% ethylene glycol effectively dissociated the complex. The dissociated DNA polymerase and primase were purified to eliminate cross-contaminating activities by column chromatography using buffers containing 50% ethylene glycol. The sedimentation coefficients of the purified DNA polymerase and primase were 7.1S and 5.7S, respectively. These two enzymes were mixed in the presence of 20% ethylene glycol and the mixture was sedimented through a glycerol gradient containing no ethylene glycol. The DNA polymerase and primase activities co-sedimented at 9.1S which corresponds to the S value of intact alpha 1, indicating the reconstitution of the DNA polymerase alpha-primase complex.

Animals↗
PubMed1991

Properties of the 3' to 5' exonuclease associated with porcine liver DNA polymerase gamma. Substrate specificity, product analysis, inhibition, and kinetics of terminal excision.

Porcine liver DNA polymerase gamma was shown previously to copurify with an associated 3' to 5' exonuclease activity (Kunkel, T. A., and Mosbaugh, D. W. (1989) Biochemistry 28, 988-995). The 3' to 5' exonuclease has now been characterized, and like the DNA polymerase activity, it has an absolute requirement for a divalent metal cation (Mg2+ or Mn2+), a relatively high NaCl and KCl optimum (150-200 mM), and an alkaline pH optimum between 7 and 10. The exonuclease has a 7.5-fold preference for single-stranded over double-stranded DNA, but it cannot excise 3'-terminal dideoxy-NMP residues from either substrate. Excision of 3'-terminally mismatched nucleotides was preferred approximately 5-fold over matched 3' termini, and the hydrolysis product from both was a deoxyribonucleoside 5'-monophosphate. The kinetics of 3'-terminal excision were measured at a single site on M13mp2 DNA for each of the 16 possible matched and mismatched primer.template combinations. As defined by the substrate specificity constant (Vmax/Km), each of the 12 mismatched substrates was preferred over the four matched substrates (A.T, T.A, C.G, G.C). Furthermore, the exonuclease could efficiently excise internally mismatched nucleotides up to 4 residues from the 3' end. DNA polymerase gamma was not found to possess detectable DNA primase, endonuclease, 5' to 3' exonuclease, RNase, or RNase H activities. The DNA polymerase and exonuclease activities exhibited dissimilar rates of heat inactivation and sensitivity to N-ethylmaleimide. After nondenaturing activity gel electrophoresis, the DNA polymerase and 3' to 5' exonuclease activities were partially resolved and detected in situ as separate species. A similar analysis on a denaturing activity gel identified catalytic polypeptides with molecular weights of 127,000, 60,000, and 32,000 which possessed only DNA polymerase gamma activity. Collectively, these results suggest that the polymerase and exonuclease activities reside in separate polypeptides, which could be derived from separate gene products or from proteolysis of a single gene product.

Animals↗
PubMed2001

A mutation in subunit B of the DNA polymerase alpha-primase complex from Novikoff hepatoma cells concomitant with a conformational change and abnormal catalytic properties of the DNA polymerase alpha-primase complex.

Mutated constituents of the DNA replication complex might contribute to the mutational load of the genome during tumor development by impairing DNA synthesis as well as cell cycle-related control of DNA replication. To prove or disprove this hypothesis, we looked for mutations in the cDNA sequences of the four subunits of DNA polymerase alpha-primase from both highly malignant Novikoff hepatoma cells and regenerating normal rat liver and compared physicochemical and catalytic properties of the DNA polymerase alpha-primase complexes purified from both sources. Sequence analysis showed two mutations in subunit B from Novikoff cells: one in nucleotide position 855 (CCG-->CCA) that did not result in an amino acid exchange and one in position 862 (GTG-->ATG) that caused a change of valine to methionine in codon 288. No mutation was found in the three other subunits. The wild-type and mutated sequences of subunit B were cloned and expressed in vitro. Sedimentation analysis of the expressed polypeptides revealed different sedimentation constants, indicating that the amino acid exchange affected the conformation of subunit B. The analysis of the purified DNA polymerase alpha-primase complexes showed a sedimentation value that was significantly higher for the enzyme complex from normal liver than for that from Novikoff cells. In addition, DNA polymerase alpha-primase complexes from Novikoff cells showed higher sensitivity to camptothecin, topotecan, and structurally related compounds (such as (R,S)-7-ethyl-10-hydroxy camptothecin, 9-aminocamptothecin, and 10-hydroxycamptothecin) than the enzyme from normal rat liver. Thus, the amino acid change found in subunit B appears to result in a conformational change of the DNA polymerase alpha-primase complex from Novikoff hepatoma cells. Whether this mutation influences genetic instability or tumor development needs to be explored.

Animals↗
PubMed1985

Inhibitory effects of various 3'-deoxyribonucleotides on DNA polymerase alpha 2-primase from developing cherry salmon (Oncorhynchus masou) testes.

DNA polymerase alpha 2-primase has been purified 2750 fold from developing cherry salmon (Oncorhynchus masou) testes by the following purification steps: fractional extraction, phosphocellulose (1st), ammonium sulfate fractionation, DEAE-cellulose, phosphocellulose (2nd), hydroxylapatite and single-stranded DNA-cellulose column chromatographies. Final preparation of this enzyme has a specific activity of 107,000 units/mg protein (activated salmon sperm DNA as template-primer). DNA primase activity (rGTP dependent incorporation of labelled dGMP into poly (dC) or rNTP dependent incorporation of dNMP into M13 single-stranded DNA) was tightly associated with DNA polymerase alpha activity during all stage of this purification process. Inhibition of DNA primase activity by six kinds of 3'-deoxyribonucleotides was studied by using rNTP dependent DNA synthesis on M13 DNA as template. The inhibition constants (Ki) were larger than those of DNA-dependent RNA polymerases I and II. However, Ki/Km values were very close.

Animals↗
PubMed1993

Purification and characterization of a yeast DNA polymerase alpha complex with associated primase, 5'-->3' exonuclease, and DNA-dependent ATPase activities.

We have purified a multimeric form of yeast DNA polymerase alpha with DNA polymerase, primase, 5'-->3' exonuclease, and single-stranded (ss) DNA-dependent ATPase activities to near-homogeneity. The molecular mass of complex was 650 kDa with subunits ranging in sizes from 30 to 180 kDa. The alpha-subunit of the complex could be detected by DNA polymerase alpha antibody. No cross-reactivity of polypeptides within the complex was observed with antibodies directed against polymerase delta or epsilon. The multimeric polymerase alpha could be selectively inhibited by p-n-butylphenyl-dGTP (I50 of approximately 0.2 microM), p-n-butylanilino-dATP (I50 of 1.3 microM), and aphidicolin (I50 of 2.5 micrograms/mL). The complex synthesized RNA primers on various ssDNA templates and rapidly elongated these primers into nascent DNA fragments in the presence of required deoxynucleotides. It has a strong 5'-->3' exonuclease activity. In addition, the complex hydrolyzed both ATP and dATP in a ssDNA-dependent manner. Thus, the multiprotein complex of DNA polymerase alpha had multiple activities (primase, polymerase, and ATPase) which could act concertedly to synthesize primers and elongate the primers to nascent DNA fragments in the lagging strand of the fork.

Adenosine Triphosphatases↗
PubMed1994

Species-specific functional interactions of DNA polymerase alpha-primase with simian virus 40 (SV40) T antigen require SV40 origin DNA.

Physical and functional interactions of simian virus 40 (SV40) and polyomavirus large-T antigens with DNA polymerase alpha-primase were analyzed to elucidate the molecular basis for the species specificity of polymerase alpha-primase in viral DNA replication. SV40 T antigen associated more efficiently with polymerase alpha-primase in crude human extracts than in mouse extracts, while polyomavirus T antigen interacted preferentially with polymerase alpha-primase in mouse extracts. The apparent species specificity of complex formation was not observed when purified polymerase alpha-primases were substituted for the crude extracts. Several functional interactions between T antigen and purified polymerase alpha-primase, including stimulation of primer synthesis and primer elongation on M13 DNA in the presence or absence of the single-stranded DNA binding protein RP-A, also proved to be independent of the species from which polymerase alpha-primase had been purified. However, the human DNA polymerase alpha-primase was specifically required for primosome assembly and primer synthesis on SV40 origin DNA in the presence of T antigen and RP-A.

Animals↗
PubMed1997

Phosphorylation of DNA polymerase alpha-primase by cyclin A-dependent kinases regulates initiation of DNA replication in vitro.

DNA polymerase alpha-primase is the only known eukaryotic enzyme that can start DNA replication de novo. In this study, we investigated the regulation of DNA replication by phosphorylation of DNA polymerase alpha-primase. The p180 and the p68 subunits of DNA polymerase alpha-primase were phosphorylated using Cyclin A-, B- and E- dependent kinases. This phosphorylation did not influence its DNA polymerase activity on activated DNA, but slightly stimulated primase activity using poly(dT) single-stranded DNA (ssDNA) without changing the product length of primers. In contrast, site-specific initiation of replication on plasmid DNA containing the SV40 origin is affected: Cyclin A-Cdk2 and Cyclin A-Cdc2 inhibited initiation of SV40 DNA replication in vitro, Cyclin B-Cdc2 had no effect and Cyclin E-Cdk2 stimulated the initiation reaction. DNA polymerase alpha-primase that was pre-phosphorylated by Cyclin A-Cdk2 was completely unable to initiate the SV40 DNA replication in vitro; Cyclin B-Cdc2-phosphorylated enzyme was moderately inhibited, while Cyclin E-Cdk2-treated DNA polymerase alpha-primase remained fully active in the initiation reaction.

CDC2-CDC28 Kinases↗