Stimulation of DNA polymerase alpha activity by microtubule-associated proteins.

Microtubule-associated protein 2 (MAP2) isolated from porcine brains stimulated the activity of DNA polymerase alpha immunopurified from calf thymus or human lymphoma cells, in a dose-dependent manner. This stimulation was pronounced when activated DNA or poly(dA).(dT)10 was used as the template-primer. DNA polymerase alpha bound to a MAP2-immobilized column, whereas preincubation of the enzyme with unbound MAP2 prevented binding to the column. These events suggested that a physical binding occurred between the polymerase and MAP2. Kinetic analyses revealed that MAP2 decreased the Km value of the polymerase for deoxyribonucleotides, irrespective of the species of template-primer. A concomitant increase in Vmax was observed; however, the extent of the increase depended on the species of template-primer. MAP2 also decreased the Km value of the polymerase for template-primers when activated DNA of poly(dA).(dT)10 was used as the template-primer. Product analyses showed that MAP2 did not significantly alter the processivity of the polymerase and the increment of Vmax is considered to be due to an increase in the frequency of initiation of DNA synthesis. The stimulation by MAP2 occurred specifically in the activity of DNA polymerase alpha, but not DNA polymerases beta, gamma, and I from Escherichia coli. Other MAPs, tau and 190-kDa MAP, could substitute for MAP2. Thus, the specific stimulation of DNA polymerase alpha by MAPs supports the notion of a possible involvement of MAPs or MAP-like proteins in DNA replication, in vivo.     

Stimulation of bull seminal RNase by various basic proteins

The activity of purified bull seminal RNase was markedly stimulated by various basic proteins. At the half concentration of substrate RNA, basic proteins such as histones, high-mobility group chromosomal proteins and cytochrome c stimulated the enzyme activity 4-6 fold. Other non-basic proteins such as bovine serum albumin and human gamma-globulin were far less effective. In addition to enzyme-stimulating activity, basic proteins showed a marked enzyme-stabilizing activity, indicating the presence of a strong interaction between the enzyme and basic proteins.     

Stimulation of DNA polymerase alpha by hypergravity generated by centrifugal acceleration.

Gravity alteration is known to influence cell proliferation. Here we tested the effects of hypergravity on the action of DNA polymerase alpha, one of the DNA replication enzymes in eukaryotes. Hypergravity was produced by horizontal centrifugal acceleration with a hand-made rotator. The reaction rate of DNA polymerase alpha in centrifuge tubes increased along with the acceleration up to 4g, when a plateau was reached. In contrast, no stimulation was observed with primase, DNA polymerase epsilon, and the E. coli DNA polymerase I Klenow fragment. Kinetic analysis of DNA polymerase alpha reactions revealed that, under high gravity conditions, the K(m) value for template DNA decreased while the V(max) stayed constant. In contrast, the centrifugal acceleration did not affect the K(m) values for deoxyribonucleoside triphosphates. These results suggest that the hypergravity enhances the activity of DNA polymerase alpha by increasing the affinity of the enzyme for template DNA. Such enhancement was more prominent with a low concentration of DNA polymerase alpha under low ionic conditions.     

Calcium-dependent calmodulin-binding proteins associated with mammalian DNA polymerase alpha

Complex, multiprotein forms of bovine (calf thymus), hamster (Chinese hamster ovary cell), and human (HeLa) cell DNA polymerase alpha (Pol alpha) were analyzed for their content of calmodulin-binding proteins. The approach used an established autoradiographic technique employing 125I-labeled calmodulin to probe proteins in denaturing SDS-polyacrylamide gel electropherograms. All three Pol alpha enzymes were associated with discrete, Ca2+-dependent calmodulin-binding proteins. Conventionally purified calf thymus Pol alpha holoenzyme contained three prominent, trifluoperazine-sensitive species with apparent molecular masses of approx. 120, 80 and 48 kDa. The 120 and 48 kDa species remained associated with the polymerase.primase core of the calf enzyme during immunopurification with monoclonal antibodies directed specifically against the polymerase subunit. The patterns of the calmodulin-binding proteins displayed by conventionally purified preparations of hamster and human Pol alpha enzymes were similar to each other and distinctly different from the pattern of comparable preparations of calf thymus Pol alpha. Immunopurified preparations of the human and hamster Pol alphas retained significant calmodulin-binding activity of apparent molecular masses of approx. 55, 80 and 150-200 kDa.     

Inhibition of DNA polymerase alpha activity by proteins from rat liver

We determined that there is a protein in rat liver capable of inhibiting DNA polymerase alpha. To assay for this inhibitor, DNA polymerase alpha was purified from R3230AC rat mammary tumor, a rich source of this enzyme. Protein fractions from Sephacryl S200 gel filtration of total soluble liver extract showing inhibition of DNA polymerase alpha were further chromatographed on DEAE-cellulose. This step revealed two inhibitor protein populations with the major form corresponding to a molecular weight of 143,000 dalton. Soluble extract from isolated rat liver nuclei also showed the presence of at least two inhibitors; the major form was 200,000+ dalton in molecular weight. Both the 143,000 and 200,000+ dalton inhibitor proteins were capable of inhibiting the R3230AC tumor DNA polymerase alpha in a dose-dependent manner. These inhibitors exhibited similar inhibition of nuclear matrix-associated DNA polymerase alpha from either the R3230AC tumor or from regenerating rat liver.     

Aphidicolin resistant mutant of which DNA polymerase alpha is induced by this drug

Mutants resistant to aphidicolin, a specific inhibitor of DNA polymerase α of eukaryotic cells, were selected from cultured FM3A cells, derived from mouse mammary carcinoma. One of them, designated as Aph 212, grew in the presence of 1 μg/ml of the drug, which did not permit wild type cells to grow. The resistance of Aph 212 cells to aphidicolin seems to be due to the increment of the activity of DNA polymerase α when Aph 212 cells were cultivated in the presence of the drug.     

Stimulation of an alpha like DNA polymerase by v-myc related protein of Halobacterium halobium

Partial DNA sequencing of a genomic clone of the archaebacterium Halobacterium halobium, which hydridized with an avian v-myc probe, showed especially the presence, in the organism of one of the conserved regions through myb, myc and adenovirus E1a oncogenes. The archaebacterial deduced amino acid sequence displayed significant homology with the v-myc gene product. In accordance with the partial DNA sequencing which assured a sufficient homology to have similar epitopes, a protein having a molecular weight of 70,000 and possessing high antigenicity with a polyclonal antiserum against avian v-myc protein was isolated and purified from H. halobium extracts. The purified v-myc like protein stimulated in vitro DNA synthesis carried out by the alpha like DNA polymerase of H. halobium.     

Selective inhibition of DNA polymerase alpha by a polysaccharide purified from slime of Physarum polycephalum

A polysaccharide was purified from the slime of a myxomycete, Physarum polycephalum, and its inhibitory effect on eukaryotic DNA polymerases was examined. Almost all the calf thymus DNA polymerase alpha activity was inhibited with higher than 0.2 mg/ml of the polysaccharide, when the assay was carried out with activated DNA as a template. The inhibitory effect occurred regardless of the amounts of the enzyme and deoxyribonucleotides, however, kinetic analysis revealed that the inhibition occurs competitively with the template DNA, the Ki value being 4 micrograms/ml. Inhibition was observed for DNA polymerase alpha, but not for DNA polymerases beta and gamma from various eukaryote species.     

The fidelity of DNA synthesis by the catalytic subunit of yeast DNA polymerase alpha alone and with accessory proteins

The fidelity of DNA synthesis catalyzed by the 180-kDa catalytic subunit (p180) of DNA polymerase alpha from Saccharomyces cerevisiae has been determined. Despite the presence of a 3'----5' exonuclease activity (Brooke et al., 1991, J. Biol. Chem., 266, 3005-3015), its accuracy is similar to several exonuclease-deficient DNA polymerases and much lower than other DNA polymerases that have associated exonucleolytic proofreading activity. Average error rates are 1/9900 and 1/12,000, respectively, for single base-substitution and minus-one nucleotide frameshift errors; the polymerase generates deletions as well. Similar error rates are observed with reactions containing the 180-kDa subunit plus an 86-kDa subunit (p86), or with these two polypeptides plus two additional subunits (p58 and p49) comprising the DNA primase activity required for DNA replication. Finally, addition of yeast replication factor-A (RF-A), a protein preparation that stimulates DNA synthesis and has single-stranded DNA-binding activity, yields a polymerization reaction with 7 polypeptides required for replication, yet fidelity remains low relative to error rates for semiconservative replication. The data suggest that neither exonucleolytic proofreading activity, the beta subunit, the DNA primase subunits nor RF-A contributes substantially to base substitution or frameshift error discrimination by the DNA polymerase alpha catalytic subunit.     

Inhibition of DNA polymerase alpha by bleomycin

Bleomycin is an important anti-tumor agent which works primarily through it's degradation of DNA template. Using synthetic single (poly[dA]-oligo-[dT]) and double stranded (poly[dA-dT]) templates, we noted significant inhibition when the BLM resistant homopolymer was used. Furthermore, when each of the components of the DNA polymerase assay were treated with bleomycin separately, followed by removal of bleomycin, significant inhibition (35%) of the enzyme was observed. The limited inhibition of DNA polymerase by BLM was attributed to residual activity of the enzyme-inhibitor complex.     

Inhibition of DNA polymerase alpha by gossypol

Our earlier studies have shown that gossypol is a specific inhibitor of DNA synthesis in cultured cells at low doses. In an attempt to determine the mechanism for the inhibition of DNA synthesis by gossypol we observed that gossypol does not interact with DNA per se but may affect some of the enzymes involved in DNA replication. These studies indicated that gossypol inhibits both in vivo and in vitro the activity of DNA polymerase alpha (EC 2.7.7.7), a major enzyme involved in DNA replication, in a time- and dose-dependent manner. Kinetic analysis revealed that gossypol acts as a noncompetitive inhibitor of DNA polymerase alpha with respect to all four deoxynucleotide triphosphates and to the activated DNA template. Inhibition of DNA polymerase alpha does not appear to be due to either metal chelation or reduction of sulfhydryl groups on the enzyme. Gossypol also inhibited HeLa DNA polymerase beta in a dose-dependent manner, but had no effect on DNA polymerase gamma. These results suggest that inhibition of DNA polymerase alpha may account in part for the inhibition of DNA synthesis and the S-phase block caused by gossypol. The data also raise the possibility that gossypol may interfere with DNA repair processes as well.     

Discontinuous DNA synthesis by purified mammalian proteins

Five proteins purified from mouse cells acting together efficiently convert a single-stranded circular DNA template to covalently closed duplex circle by a discontinuous mechanism. DNA polymerase alpha/primase with the assistance of alpha accessory factor covers the single-stranded circle with RNA-primed DNA fragments. Primers are removed by a combination of RNase H-1 and a 5'-exonuclease that was identified by its ability to complete this in vitro system. The 5'-exonuclease is required to remove residual one or two ribonucleotides at the primer/DNA junction that are resistant to RNase H-1. Gap filling is by the DNA polymerase alpha/primase, and DNA ligase I converts the DNA fragments to continuous strand. The concerted action of the five proteins emulates synthesis of the staging strand at the replication fork.     

Immunochemical studies of DNA polymerase delta: relationships with DNA polymerase alpha

A panel of murine hybridoma cell lines which produce antibodies against polypeptides present in human placental DNA polymerase delta preparations was developed. Eight of these antibodies were characterized by virtue of their ability to inhibit DNA polymerase delta activity and immunoblot the 170-kDa catalytic polypeptide. Six of these eight antibodies inhibit DNA polymerase delta but not DNA polymerase alpha, showing that the two proteins are distinct. However, the other two monoclonal antibodies inhibited both DNA polymerase delta and alpha activities, providing the first evidence that these two proteins have a structural relationship. In addition to antibodies against the catalytic polypeptide we also identified 11 antibodies which recognize 120-, 100-, 88-, 75-, 62-, 36-, and 22-kDa polypeptides in DNA polymerase delta preparations, suggesting that these proteins might be part of a replication complex. The antibody to the 36-kDa polypeptide was shown to be directed against proliferating cell nuclear antigen/cyclin. These antibodies should prove useful for studies aimed at distinguishing between DNA polymerases alpha and delta and for the investigation of the functional roles of DNA polymerase delta polypeptides.     

Regulation of DNA polymerase alpha activity by the alpha 1-adrenergic receptors in proliferatively activated rat liver cells.

The administration of the alpha 1-adrenergic antagonist prazosin to hepatectomized rats inhibited DNA synthesis induced in the remaining hepatocytes. This inhibitory effect could be reversed by the simultaneous injection of the agonist phenylephrine. In order to establish how the alpha 1-adrenergic receptors can regulate DNA replication, the effect of prazosin administration on DNA polymerase alpha was examined. At 24 h after partial hepatectomy, the activity of DNA polymerase alpha increased 5, 7 and 9 fold in the homogenates, nuclei and nuclear matrix, respectively. This increase was inhibited by 70%-80% when prazosin was injected at 1, 8 or 11 h after surgery. Kinetic studies revealed that the Km for DNA was 2 fold lower in hepatectomized than in control animals. The administration of prazosin to hepatectomized rats increased the Km to the control values. These results indicate that the alpha 1-adrenergic receptors are involved in the regulation of DNA synthesis through the activation of DNA polymerase alpha and that this activation could be produced by increasing its affinity for DNA.     

Evidence that errors made by DNA polymerase alpha are corrected by DNA polymerase delta

Eukaryotic replication begins at origins and on the lagging strand with RNA-primed DNA synthesis of a few nucleotides by polymerase alpha, which lacks proofreading activity. A polymerase switch then allows chain elongation by proofreading-proficient pol delta and pol epsilon. Pol delta and pol epsilon are essential, but their roles in replication are not yet completely defined . Here, we investigate their roles by using yeast pol alpha with a Leu868Met substitution . L868M pol alpha copies DNA in vitro with normal activity and processivity but with reduced fidelity. In vivo, the pol1-L868M allele confers a mutator phenotype. This mutator phenotype is strongly increased upon inactivation of the 3' exonuclease of pol delta but not that of pol epsilon. Several nonexclusive explanations are considered, including the hypothesis that the 3' exonuclease of pol delta proofreads errors generated by pol alpha during initiation of Okazaki fragments. Given that eukaryotes encode specialized, proofreading-deficient polymerases with even lower fidelity than pol alpha, such intermolecular proofreading could be relevant to several DNA transactions that control genome stability.     

Identification of Archaea-specific chemotaxis proteins which interact with the flagellar apparatus

Background  

Archaea share with bacteria the ability to bias their movement towards more favorable locations, a process known as taxis. Two molecular systems drive this process: the motility apparatus and the chemotaxis signal transduction system. The first consists of the flagellum, the flagellar motor, and its switch, which allows cells to reverse the rotation of flagella. The second targets the flagellar motor switch in order to modulate the switching frequency in response to external stimuli. While the signal transduction system is conserved throughout archaea and bacteria, the archaeal flagellar apparatus is different from the bacterial one. The proteins constituting the flagellar motor and its switch in archaea have not yet been identified, and the connection between the bacterial-like chemotaxis signal transduction system and the archaeal motility apparatus is unknown.     

Stimulation of human DNA polymerase epsilon by MDM2

The human DNA polymerase ε catalytic subunit consists of a 140-kDa N‐terminal domain that contains the catalytic activity and a 120-kDa C-terminal domain that binds to the other subunits and to exogenous peptides, including PCNA and MDM2. We report here that recombinant human MDM2 purified from insect cells or Escherichia coli stimulated the activity of DNA polymerase ε up to 10- and 40-fold, respectively, but not those of DNA polymerase β or Klenow fragment of E.coli DNA polymerase I. Kinetic studies indicated that MDM2 increased the maximum velocity of the reaction, but did not change substrate affinities. The stimulation depended upon the interaction of the N‐terminal 166 amino acid residues of MDM2 with the C-terminal domain of the full-length catalytic subunit, since the deletion of 166 amino acids from N‐terminal of MDM2 or the removal of the C-terminal domain of DNA polymerase ε by trypsin digestion or competition for binding to it by the addition of excess C-terminal fragment eliminated the stimulation. Since DNA polymerase ε appears to be involved in DNA replication, recombination and repair synthesis, we suggest that MDM2 binding to DNA polymerase ε might be part of a reconfiguration process that allows DNA polymerase ε to associate with repair/recombination proteins in response to DNA damage.     

Processive DNA synthesis by DNA polymerase II mediated by DNA polymerase III accessory proteins.

An interesting property of the Escherichia coli DNA polymerase II is the stimulation in DNA synthesis mediated by the DNA polymerase III accessory proteins beta,gamma complex. In this paper we have studied the basis for the stimulation in pol II activity and have concluded that these accessory proteins stimulate pol II activity by increasing the processivity of the enzyme between 150- and 600-fold. As is the case with pol III, processive synthesis by pol II requires both beta,gamma complex and SSB protein. Whereas the intrinsic velocity of synthesis by pol II is 20-30 nucleotides per s with or without the accessory proteins, the processivity of pol II is increased from approximately five nucleotides to greater than 1600 nucleotides incorporated per template binding event. The effect of the accessory proteins on the rate of replication is far greater on pol III than on pol II; pol III holoenzyme is able to complete replication of circular single-stranded M13 DNA in less than 20 s, whereas pol II in the presence of the gamma complex and beta requires approximately 5 min. We have investigated the effect of beta,gamma complex proteins on bypass of a site-specific abasic lesion by E. coli DNA polymerases I, II, and III. All three polymerases are extremely inefficient at bypass of the abasic lesion. We find limited bypass by pol I with no change upon addition of accessory proteins. pol II also shows limited bypass of the abasic site, dependent on the presence of beta,gamma complex and SSB. pol III shows no significant bypass of the abasic site with or without beta,gamma complex.