Plays an essential role in the initiation of DNA replication. During the S phase of the cell cycle, the DNA polymerase alpha complex (composed of a catalytic subunit POLA1/p180, a regulatory subunit POLA2/p70 and two primase subunits PRIM1/p49 and PRIM2/p58) is recruited to DNA at the replicative forks via direct interactions with MCM10 and WDHD1. The primase subunit of the polymerase alpha complex initiates DNA synthesis by oligomerising short RNA primers on both leading and lagging strands. These primers are initially extended by the polymerase alpha catalytic subunit and subsequently transferred to polymerase delta and polymerase epsilon for processive synthesis on the lagging and leading strand, respectively. The reason this transfer occurs is because the polymerase alpha has limited processivity and lacks intrinsic 3' exonuclease activity for proofreading error, and therefore is not well suited for replicating long complexes.
Poly(ADP-ribose) polymerase (PARP) is an element of the DNA damage surveillance network evolved by eukaryotic cells to cope with numerous environmental and endogenous genotoxic agents. PARP has been found to be involved in vivo in both cell proliferation and base excision repair of DNA. In this study the interaction between PARP and the DNA polymerase alpha-primase tetramer has been examined. We provide evidence that in proliferating cells: (i) PARP is physically associated with the catalytic subunit of the DNA polymerase alpha-primase tetramer, an association confirmed by confocal microscopy, demonstrating that both enzymes are co-localized at the nuclear periphery of HeLa cells; (ii) this interaction requires the integrity of the second zinc finger of PARP and is maximal during the S and G2/M phases of the cell cycle; (iii) PARP-deficient cells derived from PARP knock-out mice exhibited reduced DNA polymerase activity, compared with the parental cells, a reduction accentuated following exposure to sublethal doses of methylmethanesulfonate. Altogether, the present results strongly suggest that PARP participates in a DNA damage survey mechanism implying its nick-sensor function as part of the control of replication fork progression when breaks are present in the template.
Interacting selectively and non-covalently with a 4 iron, 4 sulfur (4Fe-4S) cluster; this cluster consists of four iron atoms, with the inorganic sulfur atoms found between the irons and acting as bridging ligands.
Interacting selectively and non-covalently with chromatin, the network of fibers of DNA, protein, and sometimes RNA, that make up the chromosomes of the eukaryotic nucleus during interphase.
The contributions of human DNA polymerases (pols) alpha, delta and epsilon during S-phase progression were studied in order to elaborate how these enzymes co-ordinate their functions during nuclear DNA replication. Pol delta was three to four times more intensely UV cross-linked to nascent DNA in late compared with early S phase, whereas the cross-linking of pols alpha and epsilon remained nearly constant throughout the S phase. Consistently, the chromatin-bound fraction of pol delta, unlike pols alpha and epsilon, increased in the late S phase. Moreover, pol delta neutralizing antibodies inhibited replicative DNA synthesis most efficiently in late S-phase nuclei, whereas antibodies against pol epsilon were most potent in early S phase. Ultrastructural localization of the pols by immuno-electron microscopy revealed pol epsilon to localize predominantly to ring-shaped clusters at electron-dense regions of the nucleus, whereas pol delta was mainly dispersed on fibrous structures. Pol alpha and proliferating cell nuclear antigen displayed partial colocalization with pol delta and epsilon, despite the very limited colocalization of the latter two pols. These data are consistent with models where pols delta and epsilon pursue their functions at least partly independently during DNA replication.
The contributions of human DNA polymerases (pols) alpha, delta and epsilon during S-phase progression were studied in order to elaborate how these enzymes co-ordinate their functions during nuclear DNA replication. Pol delta was three to four times more intensely UV cross-linked to nascent DNA in late compared with early S phase, whereas the cross-linking of pols alpha and epsilon remained nearly constant throughout the S phase. Consistently, the chromatin-bound fraction of pol delta, unlike pols alpha and epsilon, increased in the late S phase. Moreover, pol delta neutralizing antibodies inhibited replicative DNA synthesis most efficiently in late S-phase nuclei, whereas antibodies against pol epsilon were most potent in early S phase. Ultrastructural localization of the pols by immuno-electron microscopy revealed pol epsilon to localize predominantly to ring-shaped clusters at electron-dense regions of the nucleus, whereas pol delta was mainly dispersed on fibrous structures. Pol alpha and proliferating cell nuclear antigen displayed partial colocalization with pol delta and epsilon, despite the very limited colocalization of the latter two pols. These data are consistent with models where pols delta and epsilon pursue their functions at least partly independently during DNA replication.
We have isolated cDNA clones encoding the human DNA polymerase alpha catalytic polypeptide. Studies of the human DNA polymerase alpha steady-state mRNA levels in quiescent cells stimulated to proliferate, or normal cells compared to transformed cells, demonstrate that the polymerase alpha mRNA, like its enzymatic activity and de novo protein synthesis, positively correlates with cell proliferation and transformation. Analysis of the deduced 1462-amino-acid sequence reveals six regions of striking similarity to yeast DNA polymerase I and DNA polymerases of bacteriophages T4 and phi 29, herpes family viruses, vaccinia virus and adenovirus. Three of these conserved regions appear to comprise the functional active site required for deoxynucleotide interaction. Two putative DNA interacting domains are also identified.
Catalysis of the synthesis of a short RNA primer on a DNA template, providing a free 3'-OH that can be extended by DNA-directed DNA polymerases. Catalyzed by a DNA-directed RNA polymerase that forms a complex with alpha DNA polymerase.
Proc. Natl. Acad. Sci. U.S.A. 87, 9712-9716 (1990)[PubMed:2175912]
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.
Catalysis of the reaction: deoxynucleoside triphosphate + DNA(n) = diphosphate + DNA(n+1); the synthesis of DNA from deoxyribonucleotide triphosphates in the presence of a DNA template and primer.
J. Biol. Chem. 252, 6528-6535 (1977)[PubMed:893425]
In this report we describe the purification and structural characterization of a near homogeneous preparation of DNA polymerase-alpha that we have obtained from cultured human KB cells. When analyzed by nondenaturing gel electrophoresis, velocity gradient centrifugation, and isoelectric focusing, the enzyme activity demonstrates a constancy of gel mobility, sedimentation coefficient, and isoelectric point during the final three chromatographic steps of the purification. Native gel electrophoresis of the penultimate polymerase fraction at seven concentrations of acrylamide reveals co-migration of the enzyme activity with a single discernible protein band, at constant specific activity, and indicates that the polymerase protein is electrophoretically homogeneous at this stage. The purified enzyme has a sedimentation coefficient of 7.1 to 7.2 S at high or low ionic strength, a molecular weight gel filtration of 149,000, and an isoelectric point of pH 5.0 to 5.2. Sodium dodecyl sulfate gel analyses demonstrate that the polymerase-alpha molecule is a dimer comprised of two dissimilar subunits of 76,000 and 66,000 daltons that are present in equimolar ratio.
We have isolated cDNA clones encoding the human DNA polymerase alpha catalytic polypeptide. Studies of the human DNA polymerase alpha steady-state mRNA levels in quiescent cells stimulated to proliferate, or normal cells compared to transformed cells, demonstrate that the polymerase alpha mRNA, like its enzymatic activity and de novo protein synthesis, positively correlates with cell proliferation and transformation. Analysis of the deduced 1462-amino-acid sequence reveals six regions of striking similarity to yeast DNA polymerase I and DNA polymerases of bacteriophages T4 and phi 29, herpes family viruses, vaccinia virus and adenovirus. Three of these conserved regions appear to comprise the functional active site required for deoxynucleotide interaction. Two putative DNA interacting domains are also identified.
Evidence
3:
Inferred from Mutant PhenotypeUniProtKB
The effect of monoclonal anti-(DNA polymerase alpha) immunoglobulin G (IgG) and F(ab) fragments on DNA replication in lysolecithin-permeabilized human cells and on DNA polymerase alpha activity was determined. DNA polymerase alpha activity in vitro was inhibited equally by the same concentrations of monoclonal IgGs and F(ab) fragments. However, the IgGs and F(ab) fragments were not equally potent in inhibiting DNA replication in permeable cells. In general, the F(ab) fragments were approximately equal to 10-fold more potent than IgGs in inhibiting DNA replication, suggesting the F(ab) fragments cross the nuclear membrane more readily than IgGs. Immunocytochemical studies demonstrated that at least a fraction of anti-(DNA polymerase alpha) IgGs entered the nucleus of permeable cells. For most antibodies tested, the IgG or F(ab) concentration needed to inhibit replication was several orders of magnitude higher than that needed to neutralize polymerase alpha activity extracted from the same number of cells. Anti-(DNA polymerase alpha) F(ab) fragments were shown to inhibit the discontinuous synthesis of Okazaki DNA, as well as the maturation of Okazaki DNA to larger DNA, thereby implicating DNA polymerase alpha in both of these processes.
Interacting selectively and non-covalently with a nucleoside, a compound consisting of a purine or pyrimidine nitrogenous base linked either to ribose or deoxyribose.
Interacting selectively and non-covalently with a nucleotide, any compound consisting of a nucleoside that is esterified with (ortho)phosphate or an oligophosphate at any hydroxyl group on the ribose or deoxyribose.
The affinities of oligothymidylates and of some analogs for the template site, of a set of oligodeoxyribo- and oligoribonucleotides for the primer site, and of dNTPs and some analogs for the substrate sites of DNA polymerase I Klenow fragment and of human placenta DNA polymerase alpha were measured using them either as competitors of affinity modification or as substrates. The data obtained enable us to hypothesize that the Me2+-dependent electrostatic contact and hydrogen bond of a single internucleotide phosphate and the hydrophobic interactions of the other nucleotide units determine the formation of oligonucleotide-template site complexes. Interaction of the primer's 3'-terminal hydroxy group and of the negatively charged adjacent phosphate with the enzyme, and Watson-Crick base pairing with the template are of crucial importance for the formation of the ternary enzyme-template-primer complex. dNTP and dNMP imidazolides inactivate enzymes via an affinity modification mechanism only in the presence of the template-primer complex. dNTP affinities exceed those of dNDPs and dNMPs, the enhancement being most significant for the substrate that is complementary to the template, thus suggesting the participation of the gamma-phosphate of dNTP in the substrate selection step.
Interacting selectively and non-covalently with any protein or protein complex (a complex of two or more proteins that may include other nonprotein molecules).
Evidence
1:
Inferred from Physical InteractionUniProtKB
Structural maintenance of chromosome 1 (Smc1) is a multifunctional protein, which has been implicated in sister chromatid cohesion, DNA recombination and repair, and the activation of cell cycle checkpoints by ionizing radiation, ultraviolet light, and other genotoxic agents. In order to identify the proteins that interact with Smc1, we conducted the Tandem affinity purification (TAP) technique and analyzed the Smc1-interacting proteins via MALDI-TOF mass spectrometry. We identified minichromosome maintenance 7 (Mcm7), an essential component of the pre-replication complex, as a novel Smc1-interacting protein. Co-immunoprecipitation revealed an interaction occurring between Smc1 and Mcm7, both in vitro and in vivo. Using a GST pull-down assay, we determined that Smc1 interacts physically with Mcm7 via its N-terminal and hinge regions, and Mcm7 interacts with Smc1 via its middle region. Interestingly, we also discovered that Smc1 interacts with other DNA replication proteins, including Mcm6, RFC1, and DNA polymerase alpha. These results suggest that a functional link exists between the cohesin complex and DNA replication proteins.
Evidence
2:
Inferred from Physical InteractionUniProtKB
Poly(ADP-ribose) polymerase (PARP) is an element of the DNA damage surveillance network evolved by eukaryotic cells to cope with numerous environmental and endogenous genotoxic agents. PARP has been found to be involved in vivo in both cell proliferation and base excision repair of DNA. In this study the interaction between PARP and the DNA polymerase alpha-primase tetramer has been examined. We provide evidence that in proliferating cells: (i) PARP is physically associated with the catalytic subunit of the DNA polymerase alpha-primase tetramer, an association confirmed by confocal microscopy, demonstrating that both enzymes are co-localized at the nuclear periphery of HeLa cells; (ii) this interaction requires the integrity of the second zinc finger of PARP and is maximal during the S and G2/M phases of the cell cycle; (iii) PARP-deficient cells derived from PARP knock-out mice exhibited reduced DNA polymerase activity, compared with the parental cells, a reduction accentuated following exposure to sublethal doses of methylmethanesulfonate. Altogether, the present results strongly suggest that PARP participates in a DNA damage survey mechanism implying its nick-sensor function as part of the control of replication fork progression when breaks are present in the template.
Evidence
3:
Inferred from Physical InteractionUniProtKB
The catalytic polypeptide of DNA polymerase alpha is often observed in vitro as a family of phosphopolypeptides predominantly of 180 and 165 kDa derived from a single primary structure. The estimated Mr of this polypeptide deduced from the full-length cDNA is 165 kDa. Immunoblot analysis with polyclonal antibodies against peptides of the N- and C-termini of the deduced primary sequence indicates that the observed family of polypeptides from 180 kDa to lower molecular weight results from proteolytic cleavage from the N-terminus. Antibodies against the N-terminal peptide detect only the 180 kDa species suggesting that this higher molecular weight polypeptide may be the result of posttranslational modification of the 165 kDa primary translation product. The catalytic polypeptide is not only phosphorylated but is also found to react with lectins ConA and RCA. N-terminal sequencing of the isolated catalytic polypeptide from human cells and of the recombinant fusion proteins indicates that the often observed 165 kDa polypeptide is the in vitro proteolytic cleavage product of the modified 180 kDa protein at the specific site between lys123 and lys124 within the sequence -RNVKKLAVTKPNN-.
Evidence
4:
Inferred from Physical InteractionUniProtKB
The purified human single-stranded DNA binding protein, replication protein A (RP-A), forms specific complexes with purified SV40 large T antigen and with purified DNA polymerase alpha-primase, as shown by ELISA and a modified immunoblotting technique. RP-A associated efficiently with the isolated primase, as well as with intact polymerase alpha-primase. The 70 kDa subunit of RP-A was sufficient for association with polymerase alpha-primase. Purified SV40 large T antigen bound to intact RP-A and to polymerase-primase, but not to any of the separated subunits of RP-A or to the isolated primase. These results suggest that the specific protein-protein interactions between RP-A, polymerase-primase and T antigen may play a role in the initiating of SV40 DNA replication.
Evidence
5:
Inferred from Physical InteractionUniProtKB
Human retinoblastoma (Rb) protein, immunopurified from an extract of recombinant baculovirus infected cells, stimulated 10-100-fold the activity of DNA polymerase alpha from calf thymus or human HeLa cells. Purified Rb protein is composed of two electrophoretically distinguishable forms, i.e., partially phosphorylated and under-phosphorylated forms. Dephosphorylation of Rb protein by protein phosphatase 2A largely diminished its stimulatory effect. On the other hand, a hyperphosphorylated Rb protein, obtained from insect cells overexpressing Rb protein, cyclin E and cyclin-dependent kinase 2 simultaneously, stimulated DNA polymerase alpha more strongly than the singly-expressed Rb protein. These results indicate that the phosphorylation is crucial for the stimulation. Rb protein isolated from human Burkitt lymphoma Raji cells also stimulated DNA polymerase alpha. In contrast, Rb protein did not affect eukaryotic DNA primase or Klenow fragment of Escherichia coli DNA polymerase I. By immunoprecipitation using anti-DNA polymerase alpha antibody, Rb protein in nuclear extract of Raji cells was co-precipitated with DNA polymerase alpha. This result indicates that DNA polymerase alpha exists as a complex containing phosphorylated Rb protein in cells. DNA polymerase alpha specifically bound to a purified Rb protein-immobilized Sepharose column. Rb protein also bound to DNA polymerase alpha trapped to anti-DNA polymerase alpha antibody-Sepharose column, suggesting the direct association of these two proteins. These observations suggest a new function of phosphorylated Rb protein in the regulation of DNA replication.
Evidence
6:
Inferred from Physical InteractionUniProtKB
MSSP has been identified as a protein that binds to both single- and double-stranded sequences of a putative DNA replication origin sequence in the human c-myc gene. MSSP possesses versatile functions, including stimulation of DNA replication, transcriptional regulation, apoptosis induction, and cell transformation coordinated by c-Myc. MSSP contains two RNP domains, RNP1-A and RNP1-B, both of which are necessary for all of the functions of MSSP. In this study, we found that MSSP binds to the N-terminal region of a catalytic subunit of a human DNA polymerase alpha via its RNP domains both in vitro and in human cells. Furthermore, MSSP was released from the putative DNA replication origin of the c-myc gene after it complexed with DNA polymerase alpha, and MSSP stimulated DNA polymerase activity in vitro.
Evidence
7:
Inferred from Physical InteractionUniProtKB
Eur. J. Biochem. 265, 936-943 (1999)[PubMed:10518787]
Budding yeast CDC45 encodes Cdc45p, an essential protein required to trigger initiation of DNA replication in late G1 phase. We cloned four and one species of the human Cdc45p homolog cDNA, resulting from different splicing patterns, from HeLa cell and human placenta cDNA libraries, respectively. A comparison of the cDNAs and the genomic sequence showed that the longest encoding a 610-amino acid protein was comprised of 20 exons. One species, which lacks exon 7 and contains the shorter of two exons 18, was identical with the previously reported CDC45L cDNA and constituted 24 out of 28 clones from HeLa cells. Splicing was different in HeLa cells and TIG-1 cells, a human diploid cell line. Human CDC45 protein was found to bind directly in vitro to human minichromosome maintenance 7 protein (hMCM7) and to the p70 subunit of DNA polymerase alpha. The data support a thesis that human CDC45 acts as a molecular tether to mediate loading of the DNA polymerase alpha on to the DNA replication complex through binding to hMCM7.
We have isolated cDNA clones encoding the human DNA polymerase alpha catalytic polypeptide. Studies of the human DNA polymerase alpha steady-state mRNA levels in quiescent cells stimulated to proliferate, or normal cells compared to transformed cells, demonstrate that the polymerase alpha mRNA, like its enzymatic activity and de novo protein synthesis, positively correlates with cell proliferation and transformation. Analysis of the deduced 1462-amino-acid sequence reveals six regions of striking similarity to yeast DNA polymerase I and DNA polymerases of bacteriophages T4 and phi 29, herpes family viruses, vaccinia virus and adenovirus. Three of these conserved regions appear to comprise the functional active site required for deoxynucleotide interaction. Two putative DNA interacting domains are also identified.
The process of restoring DNA after damage. Genomes are subject to damage by chemical and physical agents in the environment (e.g. UV and ionizing radiations, chemical mutagens, fungal and bacterial toxins, etc.) and by free radicals or alkylating agents endogenously generated in metabolism. DNA is also damaged because of errors during its replication. A variety of different DNA repair pathways have been reported that include direct reversal, base excision repair, nucleotide excision repair, photoreactivation, bypass, double-strand break repair pathway, and mismatch repair pathway.
Negative evidence
1:
Inferred from Direct AssayUniProtKB
J. Biol. Chem. 263, 501-510 (1988)[PubMed:3335506]
When UV-irradiated cultured diploid human fibroblasts were permeabilized with Brij-58 then separated from soluble material by centrifugation, conservative DNA repair synthesis could be restored by a soluble factor obtained from the supernatant of similarly treated HeLa cells. Extensive purification of this factor yielded a 10.2 S, 220,000-dalton polypeptide with the DNA polymerase and 3'- to 5'-exonuclease activities reported for DNA polymerase delta II (Crute, J. J., Wahl, A. F., and Bambara, R. A. (1986) Biochemistry 25, 26-36). Monoclonal antibody to KB cell DNA polymerase alpha, while binding to HeLa DNA polymerase alpha, did not bind to the HeLa DNA polymerase delta. Moreover, at micromolar concentrations N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP) and 2-(p-n-butylanilino)-2'-deoxyadenosine 5'-triphosphate (BuAdATP) were potent inhibitors of DNA polymerase alpha, but did not inhibit the DNA polymerase delta. Neither purified DNA polymerase alpha nor beta could promote repair DNA synthesis in the permeabilized cells. Furthermore, under conditions which inhibited purified DNA polymerase alpha by greater than 90%, neither monoclonal antibodies to DNA polymerase alpha, BuPdGTP, nor BuAdATP was able to inhibit significantly the DNA repair synthesis mediated by the DNA polymerase delta. Thus, it appears that a major portion of DNA repair synthesis induced by UV irradiation might be catalyzed by DNA polymerase delta. When xeroderma pigmentosum human diploid fibroblasts were utilized, DNA repair synthesis dependent upon ultraviolet light could be restored by addition of both T4 endonuclease V and DNA polymerase delta, but not by addition of either one alone. This result suggests that cytosol-depleted permeabilized DNA repair-defective human fibroblasts and HeLa DNA polymerase delta might be exploited to provide a functional assay for purifying active DNA repair factors from DNA repair-proficient cells without a preknowledge of their function.
The cellular metabolic process in which a cell duplicates one or more molecules of DNA. DNA replication begins when specific sequences, known as origins of replication, are recognized and bound by initiation proteins, and ends when the original DNA molecule has been completely duplicated and the copies topologically separated. The unit of replication usually corresponds to the genome of the cell, an organelle, or a virus. The template for replication can either be an existing DNA molecule or RNA.
Evidence
1:
Inferred from Mutant PhenotypeUniProtKB
The contributions of human DNA polymerases (pols) alpha, delta and epsilon during S-phase progression were studied in order to elaborate how these enzymes co-ordinate their functions during nuclear DNA replication. Pol delta was three to four times more intensely UV cross-linked to nascent DNA in late compared with early S phase, whereas the cross-linking of pols alpha and epsilon remained nearly constant throughout the S phase. Consistently, the chromatin-bound fraction of pol delta, unlike pols alpha and epsilon, increased in the late S phase. Moreover, pol delta neutralizing antibodies inhibited replicative DNA synthesis most efficiently in late S-phase nuclei, whereas antibodies against pol epsilon were most potent in early S phase. Ultrastructural localization of the pols by immuno-electron microscopy revealed pol epsilon to localize predominantly to ring-shaped clusters at electron-dense regions of the nucleus, whereas pol delta was mainly dispersed on fibrous structures. Pol alpha and proliferating cell nuclear antigen displayed partial colocalization with pol delta and epsilon, despite the very limited colocalization of the latter two pols. These data are consistent with models where pols delta and epsilon pursue their functions at least partly independently during DNA replication.
Evidence
2:
Inferred from Mutant PhenotypeUniProtKB
The effect of monoclonal anti-(DNA polymerase alpha) immunoglobulin G (IgG) and F(ab) fragments on DNA replication in lysolecithin-permeabilized human cells and on DNA polymerase alpha activity was determined. DNA polymerase alpha activity in vitro was inhibited equally by the same concentrations of monoclonal IgGs and F(ab) fragments. However, the IgGs and F(ab) fragments were not equally potent in inhibiting DNA replication in permeable cells. In general, the F(ab) fragments were approximately equal to 10-fold more potent than IgGs in inhibiting DNA replication, suggesting the F(ab) fragments cross the nuclear membrane more readily than IgGs. Immunocytochemical studies demonstrated that at least a fraction of anti-(DNA polymerase alpha) IgGs entered the nucleus of permeable cells. For most antibodies tested, the IgG or F(ab) concentration needed to inhibit replication was several orders of magnitude higher than that needed to neutralize polymerase alpha activity extracted from the same number of cells. Anti-(DNA polymerase alpha) F(ab) fragments were shown to inhibit the discontinuous synthesis of Okazaki DNA, as well as the maturation of Okazaki DNA to larger DNA, thereby implicating DNA polymerase alpha in both of these processes.
Evidence
3:
Inferred from Mutant PhenotypeUniProtKB
J. Biol. Chem. 260, 134-138 (1985)[PubMed:3917431]
Monoclonal neutralizing antibodies against DNA polymerase-alpha substantially inhibit nuclear DNA replication in lysolecithin-permeabilized cultured human fibroblasts. The degree of inhibition of DNA synthesis is proportional to antibody concentration, and the effect is specific in that RNA synthesis measured under the same experimental conditions is unperturbed. Autoradiographic data demonstrate that the magnitude of the inhibition measured in the mass culture reflects the uniform response of all the constituent cells in the target population. These observations confirm the participation of DNA polymerase-alpha in replicative DNA synthesis and identify a versatile, novel approach to the dissection of mammalian processes of DNA replication and repair.
The process in which DNA-dependent DNA replication is started; this involves the separation of a stretch of the DNA double helix, the recruitment of DNA polymerases and the initiation of polymerase action.
Proc. Natl. Acad. Sci. U.S.A. 87, 9712-9716 (1990)[PubMed:2175912]
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.
The synthesis of a short RNA polymer, usually 4-15 nucleotides long, using one strand of unwound DNA as a template; the RNA then serves as a primer from which DNA polymerases extend synthesis.
Proc. Natl. Acad. Sci. U.S.A. 87, 9712-9716 (1990)[PubMed:2175912]
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.
The process in which a DNA strand is synthesized from template DNA during replication by the action of polymerases, which add nucleotides to the 3' end of the nascent DNA strand.
Evidence
1:
Inferred from Mutant PhenotypeUniProtKB
The effect of monoclonal anti-(DNA polymerase alpha) immunoglobulin G (IgG) and F(ab) fragments on DNA replication in lysolecithin-permeabilized human cells and on DNA polymerase alpha activity was determined. DNA polymerase alpha activity in vitro was inhibited equally by the same concentrations of monoclonal IgGs and F(ab) fragments. However, the IgGs and F(ab) fragments were not equally potent in inhibiting DNA replication in permeable cells. In general, the F(ab) fragments were approximately equal to 10-fold more potent than IgGs in inhibiting DNA replication, suggesting the F(ab) fragments cross the nuclear membrane more readily than IgGs. Immunocytochemical studies demonstrated that at least a fraction of anti-(DNA polymerase alpha) IgGs entered the nucleus of permeable cells. For most antibodies tested, the IgG or F(ab) concentration needed to inhibit replication was several orders of magnitude higher than that needed to neutralize polymerase alpha activity extracted from the same number of cells. Anti-(DNA polymerase alpha) F(ab) fragments were shown to inhibit the discontinuous synthesis of Okazaki DNA, as well as the maturation of Okazaki DNA to larger DNA, thereby implicating DNA polymerase alpha in both of these processes.
The human single-stranded DNA binding protein (HSSB/RPA) is involved in several processes that maintain the integrity of the genome including DNA replication, homologous recombination, and nucleotide excision repair of damaged DNA. We report studies that analyze the role of HSSB in DNA repair. Specific protein-protein interactions appear to be involved in the repair function of HSSB, since it cannot be replaced by heterologous single-stranded DNA binding proteins. Anti-HSSB antibodies that inhibit the ability of HSSB to stimulate DNA polymerase alpha also inhibit repair synthesis mediated by human cell-free extracts. However, antibodies that neutralize DNA polymerase alpha do not inhibit repair synthesis. Repair is sensitive to aphidicolin, suggesting that DNA polymerase epsilon or delta participates in nucleotide excision repair by cell extracts. HSSB has a role other than generally stimulating synthesis by DNA polymerases, as it does not enhance the residual damage-dependent background synthesis displayed by repair-deficient extracts from xeroderma pigmentosum cells. Significantly, when damaged DNA is incised by the Escherichia coli UvrABC repair enzyme, human cell extracts can carry out repair synthesis even when HSSB has been neutralized with antibodies. This suggests that HSSB functions in an early stage of repair, rather than exclusively in repair synthesis. A model for the role of HSSB in repair is presented.
Negative evidence
2:
Inferred from Mutant PhenotypeUniProtKB
Ultraviolet-induced DNA repair synthesis was measured in saponin-permeabilized normal human fibroblasts by the incorporation of [alpha-32P]dTMP into DNA. The involvement of DNA polymerases alpha, beta, delta, and epsilon in excision repair of pyrimidine dimers was examined using specific inhibitors. Dose-response curves resulting from experiments with up to 12 different inhibitor concentrations were analyzed by linear regression. Inhibitor concentrations at which repair activity was reduced to 50% were calculated. The following K50 values were found: aphidicolin, 0.2 microM; ddTTP, 12.5 microM; butylphenyl-dGTP, 7.6 microM; butylanilino-dATP, 6.0 microM. Comparison of K50 values with in vitro Ki values of DNA polymerases revealed that in permeabilized human fibroblasts reparative DNA synthesis is catalyzed by DNA polymerase delta and by DNA polymerase epsilon.
The repair of a double-strand break in DNA in which the two broken ends are rejoined with little or no sequence complementarity. Information at the DNA ends may be lost due to the modification of broken DNA ends.
Evidence
1:
Inferred from Mutant PhenotypeUniProtKB
In mammalian cells, DNA double-strand breaks are repaired mainly by non-homologous end joining, which modifies and ligates two DNA ends without requiring extensive base pairing interactions for alignment. We investigated the role of DNA polymerases in DNA-PK-dependent end joining of restriction-digested plasmids in vitro and in vivo. Rejoining of DNA blunt ends as well as those with partially complementary 5' or 3' overhangs was stimulated by 20-53% in HeLa cell-free extracts when dNTPs were included, indicating that part of the end joining is dependent on DNA synthesis. This DNA synthesis-dependent end joining was sensitive to aphidicolin, an inhibitor of alpha-like DNA polymerases. Furthermore, antibodies that neutralize the activity of DNA polymerase alpha were found to strongly inhibit end joining in vitro, whereas neutralizing antibodies directed against DNA polymerases beta and epsilon did not. DNA sequence analysis of end joining products revealed two prominent modes of repair, one of which appeared to be dependent on DNA synthesis. Identical products of end joining were recovered from HeLa cells after transfection with one of the model substrates, suggesting that the same end joining mechanisms also operate in vivo. Fractionation of cell extracts to separate PCNA as well as depletion of cell extracts for PCNA resulted in a moderate but significant reduction in end joining activity, suggesting a potential role in a minor repair pathway.
The synthesis of DNA from a template strand in a net 3' to 5' direction. Lagging strand DNA elongation proceeds by discontinuous synthesis of short stretches of DNA, known as Okazaki fragments, from RNA primers; these fragments are then joined by DNA ligase. Although each segment of nascent DNA is synthesized in the 5' to 3' direction, the overall direction of lagging strand synthesis is 3' to 5', mirroring the progress of the replication fork.
Complete enzymatic replication of DNA from the simian virus 40 origin has been reconstituted with T antigen and highly purified cellular proteins. DNA polymerase-alpha/primase functions primarily to synthesize RNA-DNA primers for initiation of DNA replication at the origin and for priming each Okazaki fragment. A polymerase switching mechanism requiring replication factor C and the proliferating cell nuclear antigen allows two molecules of DNA polymerase-delta to replicate both strands of the double helix conjointly.
Proc. Natl. Acad. Sci. U.S.A. 87, 9712-9716 (1990)[PubMed:2175912]
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.
The synthesis of DNA from a template strand in the 5' to 3' direction; leading strand elongation is continuous as it proceeds in the same direction as the replication fork.
Proc. Natl. Acad. Sci. U.S.A. 87, 9712-9716 (1990)[PubMed:2175912]
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.
Complete enzymatic replication of DNA from the simian virus 40 origin has been reconstituted with T antigen and highly purified cellular proteins. DNA polymerase-alpha/primase functions primarily to synthesize RNA-DNA primers for initiation of DNA replication at the origin and for priming each Okazaki fragment. A polymerase switching mechanism requiring replication factor C and the proliferating cell nuclear antigen allows two molecules of DNA polymerase-delta to replicate both strands of the double helix conjointly.
Repair of the gap in the DNA helix by DNA polymerase and DNA ligase after the portion of the strand containing the lesion has been removed by pyrimidine-dimer repair enzymes.
S phase occurring as part of the mitotic cell cycle. S phase is the part of the cell cycle during which DNA synthesis takes place. A mitotic cell cycle is one which canonically comprises four successive phases called G1, S, G2, and M and includes replication of the genome and the subsequent segregation of chromosomes into daughter cells.
The contributions of human DNA polymerases (pols) alpha, delta and epsilon during S-phase progression were studied in order to elaborate how these enzymes co-ordinate their functions during nuclear DNA replication. Pol delta was three to four times more intensely UV cross-linked to nascent DNA in late compared with early S phase, whereas the cross-linking of pols alpha and epsilon remained nearly constant throughout the S phase. Consistently, the chromatin-bound fraction of pol delta, unlike pols alpha and epsilon, increased in the late S phase. Moreover, pol delta neutralizing antibodies inhibited replicative DNA synthesis most efficiently in late S-phase nuclei, whereas antibodies against pol epsilon were most potent in early S phase. Ultrastructural localization of the pols by immuno-electron microscopy revealed pol epsilon to localize predominantly to ring-shaped clusters at electron-dense regions of the nucleus, whereas pol delta was mainly dispersed on fibrous structures. Pol alpha and proliferating cell nuclear antigen displayed partial colocalization with pol delta and epsilon, despite the very limited colocalization of the latter two pols. These data are consistent with models where pols delta and epsilon pursue their functions at least partly independently during DNA replication.
The replication of damaged DNA by synthesis across a lesion in the template strand; a specialized DNA polymerase or replication complex inserts a defined nucleotide across from the lesion which allows DNA synthesis to continue beyond the lesion. This process can be mutagenic depending on the damaged nucleotide and the inserted nucleotide.
Protein involved in DNA replication, i.e. the duplication of DNA by making a new copy of an existing molecule. The parental double- stranded DNA molecule is replicated semi conservatively, i.e. each copy contains one of the original strands paired with a newly synthesized strand that is complementary in terms of AT and GC base pairing.
Viral protein involved in a direct and specific interaction with a host macromolecule. Viruses interact with many cellular pathways to achieve their replication cycle. Entry into the host cell, transport to the viral replication sites or viral budding are all steps that require interaction between the host and the virus. Additionally, the evasion from the host immune response requires a lot of viral proteins to associate with and inhibit cellular proteins with antiviral functions.
Enzyme that catalyzes DNA synthesis by addition of deoxyribonucleotide units to a DNA chain using DNA as a template. They can also possess exonuclease activity and therefore function in DNA repair.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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