Characteristics of deoxyribonucleic acid polymerase activity in nuclear and supernatant fractions of cultured mouse cells

1. DNA polymerase activity is present in both nuclear and supernatant fractions prepared from rapidly dividing L929 mouse cells. 2. Nuclear preparations are 2-5 times more active with added native DNA as template and the supernatant fractions show an equivalent preference for heat-denatured DNA. 3. Isolated nuclei can carry on limited DNA synthesis in the absence of added template but are stimulated five- to ten-fold by addition of 50mug of native DNA per assay. 4. DNA polymerase activity can be released from intact nuclei by ultrasonic treatment or by extraction with 1.5m-potassium chloride. 5. The activities in nuclear and supernatant fractions, with their preferred templates, respond similarly to changes in pH and Mg(2+) and K(+) concentrations. 6. Maximal enzyme activity is approached with 40mug of DNA per assay and activation of the DNA template by treatment with deoxyribonuclease does not decrease the amount of DNA required to reach saturation. 7. The nuclear enzyme, incubated with native DNA, is markedly inhibited by the addition of heat-denatured DNA to the assay. In contrast, the supernatant DNA polymerase activity on denatured templates is not affected by the presence of native DNA. 8. The nuclear enzyme exhibits high activity in the absence of one or more deoxyribonucleoside triphosphates but this is much diminished after partial purification of the enzyme by precipitation at pH5 and fractionation on Sephadex G-200 columns. 9. The (3)H-labelled DNA products formed by Sephadex-purified nuclear and supernatant fractions, with their preferred templates, were found to be resistant to treatment with exonuclease I. Alkali-denaturation of the (3)H-labelled DNA products rendered them susceptible to attack by exonuclease I. 10. Analysis of the products on alkaline sucrose density gradients suggests that the newly synthesized material may not be covalently bound to the original DNA template. 11. By using their preferred templates the specific activity of supernatant fractions varies markedly with the position of the cells in the cell-cycle, but the specific activity of nuclear fractions varies only slightly.     

Reconstitution of endogenous DNA synthesis in isolated nuclei and template activity of chromatin with respect to mode of inhibition by aphidicolin

We succeeded in reconstituting the endogenous nuclear DNA synthesis of the sea urchin. Endogenous DNA synthesis of isolated nuclei was reconstituted by mixing the salt-treated nuclei (chromatin exhibiting essentially no endogenous DNA synthesis) and the salt extract containing DNA polymerase-alpha. DNA synthesis in this reconstitution system showed a level of activity and a mode of inhibition by aphidicolin similar to those of the original isolated nuclei (noncompetitive with respect to dCTP). On the other hand, the inhibitory mode was competitive with respect to dCTP in DNA synthesis in the reconstituted system obtained from the chromatin and purified DNA polymerase-alpha, indicating that some other factor(s) in addition to DNA polymerase-alpha is necessary for the reconstitution with reference to the inhibitory mode of aphidicolin. We also studied the template activity of the chromatin. When chromatin was used as a template, inhibition by aphidicolin of DNA polymerase-alpha was noncompetitive and uncompetitive with respect to the template at high and low concentrations, respectively. Treatment of chromatin with 5 M urea gave urea-treated chromatin (nonhistone protein-deprived chromatin) and the extract (mainly nonhistone protein fraction). Inhibition by aphidicolin of DNA polymerase-alpha was uncompetitive with respect to the urea-treated chromatin. However, when chromatin reconstituted from the urea-treated chromatin and the extract was used as a template, the inhibitory mode by aphidicolin was similar to that with original chromatin, indicating that the nonhistone protein fraction contained factor(s) which modified the inhibitory mode of aphidicolin. Thus, the inhibitory mode of aphidicolin is a useful parameter for monitoring the resolution and reconstitution of endogenous DNA synthesis of isolated nuclei.     

Studies on histone oligomers. V. Reconstitution of chromatin from purified DNA and acid-extracted histones

DNA-histone complexes were reconstituted from DNA and acid-extracted core histones and the products were characterized by micrococcal nuclease digestion to examine whether proper nucleosome structure had been reconstituted. No nucleosome structure was produced starting from the mixture of acid-extracted histones and purified DNA in 2 M NaCl-5 M urea, while the reassociation of chromatin by the same procedures was successful. This was due to the inappropriate conformation of acid-extracted histones, which was preserved in 2 M NaCl even in the presence of 5 M urea. If acid-extracted histones were reannealed from the completely denatured state, such as in 5 M urea, 6 M guanidine hydrochloride or 0.6 M NaCl-5 M urea, reconstitution of nucleosome structure was always successful.     

Identification and Characterization of Two DNA Polymerase Activities Present in Trypanosoma brucei Mitochondria

We have identified and partially purified two DNA polymerase activities from purified Trypanosoma brucei mitochondrial extracts. The DNA polymerase activity eluted from the single-stranded DNA agarose column at 0.15 M KCI (polymerase MI) was significantly inhibited by salt concentrations greater than 100 mM, utilized Mg²⁺ in preference to Mn²⁺ as a cofactor on deoxyribonucleotide templates with deoxyribose primers, and in the presence of Mn²⁺ favored a ribonucleotide template with a deoxyribose primer. A 44 kDa peptide in this fraction crossreacted with antisera against the Crithidia fasciculata β-like mitochondrial polymerase. In activity gels the catalytic peptide migrated at an apparent molecular weight of 35 kDa. The DNA polymerase activity present in the 0.3 M KCI DNA agarose fraction (polymerase M2) exhibited optimum activity at 120-180 mM KCI, used both Mg²⁺ and Mn²⁺ as cofactors, and used deoxyribonucleotide templates primed with either deoxyribose or ribose oligomers. Activity gel assays indicate that the native catalytic peptide(s) is ˜ 80 kDa in size. The two polymerases showed different sensitivities to several inhibitors: polymerase MI shows similarities to the Crithidia fasciculata β-like mitochondrial polymerase while polymerase M2 is a novel, salt-activated enzyme of higher molecular weight.     

Influence of template strandedness on in vitro replication of mutagen-damaged DNA

We analyzed the ability of DNA polymerases to bypass damage on single- and double-stranded templates. In vitro DNA synthesis was studied on UV-irradiated and polyaromatic hydrocarbon reacted (benzo[a]pyrenediol epoxide and oxiranylpyrene) double-stranded templates by a protocol involving initiation on a uniquely nicked circular double-stranded template. The template was prepared by treating single-stranded (+)M13mp2 circular strands with mutagen and then hybridizing with restricted M13 RFmp2, followed by isolation of the nicked RFII forms. The protocol permits either (+), (-), or both strands to carry lesions. We found that the rules for termination and bypass of lesions previously observed with single-stranded DNA templates also hold for double-stranded templates. Termination of synthesis occurs primarily one nucleotide 3' to the lesion in the template strand. Bypass of UV-induced lesions can be followed in a series of three partial reactions in the presence of Mn2+ and dGMP, which relax the specificity of nucleotide insertion and 3'----5' exonuclease activity, respectively. There is no evidence for greater permissivity of bypass in double-as opposed to single-stranded templates. As with single-stranded templates, purines and preferentially deoxyadenosine (dA) are inserted opposite lesions. Lesions in the nontemplate strand elicit neither termination nor pausing. The addition of Rec A protein resulted in a measurable increase of bypass in this system.     

Template-selective stimulation of diverse DNA polymerases by the murine DNA-binding protein factor D

Factor D, a template-selective DNA polymerase-alpha stimulatory protein from mouse liver (Fry, M., Lapidot, J., and Weisman-Shomer, P. (1985) Biochemistry 24, 7549-7556) is shown here to enhance the activities of diverse DNA polymerases with a cognate template specificity. DNA synthesis catalyzed by Escherichia coli DNA polymerase I, avian myeloblastosis virus polymerase, and some mammalian alpha- and gamma-polymerases was increased by factor D. With every enhanced polymerase, factor D increased the rate of copying of only poly(dT) among various tested synthetic poly-deoxynucleotides. Of the natural DNA templates examined, rates of copying of sparsely primed denatured DNA and of singly primed circular phi X174 or M13 bacteriophage DNA, but not of activated DNA, were enhanced. Michaelis constants (Km) of affected templates with responsive polymerases were decreased by factor D, without alteration in maximum velocity (Vmax). By contrast, factor D increased Vmax of deoxyribonucleoside 5'-monophosphate incorporation without changing Km of deoxyribonucleoside 5'-triphosphate substrates. Binding of factor D to poly(dT), poly(dA).poly(dT), and DNA, but less to poly(dA), was indicated by specific retention of their complexes on a DEAE-cellulose column. That factor D does not bind to DNA polymerase-alpha or to its complex with the DNA template was demonstrated by the failure of the factor to be coprecipitated with alpha-polymerase by anti-polymerase-alpha monoclonal antibodies in either the absence or presence of various templates. Lack of binding of factor D to the polymerase molecule was also indicated by simultaneous maximum stimulation of two competing polymerases by a limiting amount of factor. These combined results suggest that the enhancement of DNA synthesis is exerted through interaction of factor D with the template. It is proposed that this association leads to a tighter binding of the polymerase to the template and facilitates DNA synthesis.     

Interaction of DNA polymerase I of Escherichia coli with nucleotides. Antagonistic effects of single-stranded polynucleotide homopolymers

Binding of deoxyribonucleoside 5'-triphosphates to DNA polymerase I of Escherichia coli was measured by using a microscale nonequilibrium dialysis method. It allowed rapid and economic measurement of dissociation constants, with negligible interfering side reactions. A stoichiometry of 1 mol of nucleoside 5'-triphosphate/mol of DNA polymerase was measured, and the occurrence of a single binding site was established, for which the nucleotides competed in the binary complex with the polymerase. Binding affinities decreased in the order dGTP greater than or equal to dATP greater than dCTP congruent to dTTP. These results are in agreement with previous findings [Englund, P. T., Huberman, J. A., Jovin, T. M., & Kornberg, A. (1969) J. Biol. Chem. 244, 3038-3044] except that, in a few cases, values of dissociation constants were smaller by factors of 2-3. The cations Mg2+ and Mn2+, as well as spermine, slightly enhanced complex stability at low levels and decreased it at high concentrations, while NaCl and Hg2+ had only destabilizing effects. Recognition between nucleoside 5'-triphosphates and nucleotide templates was studied by titration of the polymerase-[3H]dGTP complex with polynucleotide homopolymers. Complementary poly(dC) did not affect binding of dGTP, and non-complementary templates caused rejection of the nucleotide. Rejection of dGTP followed a saturation dependence with an equivalence of 110 +/- 10 monomer units of polynucleotides bound per molecule of DNA polymerase. The results favor a model by which recognition arises chiefly from the stereogeometrical fit of complementary template and nucleoside 5'-triphosphate into a rigid binding site.     

Characterization of an RNA-directed DNA polymerase found in association with murine intracytoplasmic A-particles.

An RNA-directed DNA polymerase was found to be associated with intracytoplasmic A-particles from DBA/2 mouse leukemia cells. The enzyme activity was detected after disrupting the purified particles with 2 M NaCl-20 mM dithiothreitol. The presence of a divalent cation and all four deoxyribonucleoside triphosphates was essential for this enzyme activity. The enzyme had a clear preference for Mg2+ over Mn2+. Cesium sulfate isopycnic gradient centrifugation of the DNA product synthesized in the actinomycin D-containing reaction revealed the presence of DNA-RNA hybrid. Furthermore, the purified DNA product was found to hybridize with RNA isolated from A-particles. These observations strongly indicate that the endogenous A-particle RNA serves as the template for the DNA polymerase.     

Contrasting effects of Escherichia coli single-stranded DNA binding protein on synthesis by T7 DNA polymerase and Escherichia coli DNA polymerase I (large fragment). Evidence that binding protein inhibits trans-lesion synthesis by polymerase I

The effect of Escherichia coli single-stranded DNA binding protein (SSB) on DNA synthesis by T7 DNA polymerase and E. coli DNA polymerase I (large fragment) using native or aminofluorene-modified M13 templates was evaluated by in vitro DNA synthesis assays and polyacrylamide gel electrophoresis analysis. The two polymerase enzymes displayed differential responses to the addition of SSB. T7 DNA polymerase, a enzyme required for the replication of the T7 chromosome, was stimulated by the addition of SSB whether native or modified templates were used. On the other hand, E. coli DNA polymerase I was slightly stimulated by the addition of SSB to the native template but substantially inhibited on modified templates. This result suggests that DNA polymerase I may be able to synthesize past an aminofluorene adduct but that the presence of SSB inhibited this trans-lesion synthesis. Polyacrylamide gels of the products of DNA synthesis by polymerase I supported this inference since SSB caused a substantial increase in the accumulation of shorter DNA chains induced by blockage at the aminofluorene adduct sites.     

Rabbit liver factor D, a poly(thymidine) template stimulatory protein of DNA polymerases: purification and characterization

Factor D, a DNA binding protein that enhances the activities of diverse DNA polymerases with a common restricted set of templates, was initially characterized in mouse liver but has resisted extensive purification. In this paper, we report that a similar stimulatory activity can be obtained in highly purified form from nuclei of rabbit hepatocytes. The rabbit liver protein increases the rates at which several DNA polymerases copy sparsely primed natural DNA templates and primed synthetic poly(dT), but it has no effect on the rates of copying of activated DNA or of poly(dG), poly(dA), and poly(dC). Direct binding of the purified stimulatory protein to an oligomer that contains a (dT)16 base stretch is visualized by retardation of the nucleoprotein complex on nondenaturing electrophoretograms. In the presence of the enhancing factor, Michaelis constants, Km, of responsive polymerase for singly primed bacteriophage M13 DNA and for poly(dT), but not for poly(dA), are decreased. Product analysis of M13 DNA primer extension indicates that the rabbit factor augments the apparent processivity of DNA polymerase by decreasing the extent of enzyme pausing at a tract of four consecutive thymidine residues in the template. Gel filtration of the native stimulatory protein yields an apparent relative molecular size of 58 +/- 2 kilodaltons. Stimulatory activity is readily inactivated by heat or by trypsin digestion, but it is resistant to micrococcal nuclease, N-ethyl-maleimide, or calcium ions.     

The functional role of a DNA primase in chloroplast DNA replication in Chlamydomonas reinhardtii.

A complementation experiment was developed to identify the protein component that is essential for the in vitro replication of a cloned template containing a chloroplast DNA replication origin of Chlamydomonas reinhardtii. Using this method, we have identified a DNA primase activity that copurified with DNA polymerase from the crude protein mixture. The primase catalyzed the synthesis of short RNA primers on single-stranded DNA templates. Among the synthetic templates, the order of preference was poly(dA), poly(dT), and poly(dC). The primer size range for these templates was 11-18, 5-12, and 3-11 nucleotides, respectively. On a single-stranded template containing the chloroplast DNA replication origin, the primer length range reached 19 to 27 nucleotides, indicating a better processtivity. Several initiation sites were mapped on both strands of the cloned replication origin. Some preferential initiation sites were located on A tracks spaced at one helical turn apart within the bending locus. Primase improved the template specificity of the in vitro DNA replication system and enhanced the incorporation of radioactive dATP into the supercoiled template containing the core sequences of the chloroplast DNA replication origin.     

Sea urchin sperm DNP. I. Chemical composition and template properties of DNP

Electrophoretic mobility, amino acid composition and salt dissociation of histones isolated from sperm of sea urchin Strongylocentrotus intermedius and calf thymus cells were studied. The special arginine-rich histone fraction (I) has been observed in sea urchin sperm chromatin, this fraction being absent in calf thymus chromatin. Dissociation of lysine-containing histone fractions from sea urchin chromatin occured in the range of 0.7 to 1.0 M NaCl concentrations. H1 of calf thymus chromatin was totally extracted with 0.6 M NaCl. In the course of a further increase of salt concentrations (up to 1.5 M NaCl) a practically total extraction of histones from sperm chromatin was observed, while about 20% of proteins remained bound to DNA in thymus chromatin after extraction with 2.0 M NaCl. The template activity of non-extracted DNP preparations from urchin sperm was equal to 2-3% of that of totally deproteinized DNA. The template activity of DNP gradually increased at protein extraction from DNP preparations. The hybridization capacity of RNA transcribed on partially dehistonized DNP templates in vitro also increased.     

Insertion of dNTPs opposite the 1,N2-propanodeoxyguanosine adduct by Sulfolobus solfataricus P2 DNA polymerase IV

1, N (2)-Propanodeoxyguanosine (PdG) is a stable structural analogue for the 3-(2'-deoxy-beta- d- erythro-pentofuranosyl)pyrimido[1,2-alpha]purin-10(3 H)-one (M 1dG) adduct derived from exposure of DNA to base propenals and to malondialdehyde. The structures of ternary polymerase-DNA-dNTP complexes for three template-primer DNA sequences were determined, with the Y-family Sulfolobus solfataricus DNA polymerase IV (Dpo4), at resolutions between 2.4 and 2.7 A. Three template 18-mer-primer 13-mer sequences, 5'-d(TCACXAAATCCTTCCCCC)-3'.5'-d(GGGGGAAGGATTT)-3' (template I), 5'-d(TCACXGAATCCTTCCCCC)-3'.5'-d(GGGGGAAGGATTC)-3' (template II), and 5'-d(TCATXGAATCCTTCCCCC)-3'.5'-d(GGGGGAAGGATTC)-3' (template III), where X is PdG, were analyzed. With templates I and II, diffracting ternary complexes including dGTP were obtained. The dGTP did not pair with PdG, but instead with the 5'-neighboring template dC, utilizing Watson-Crick geometry. Replication bypass experiments with the template-primer 5'-TCACXAAATCCTTACGAGCATCGCCCCC-3'.5'-GGGGGCGATGCTCGTAAGGATTT-3', where X is PdG, which includes PdG in the 5'-CXA-3' template sequence as in template I, showed that the Dpo4 polymerase inserted dGTP and dATP when challenged by the PdG adduct. For template III, in which the template sequence was 5'-TXG-3', a diffracting ternary complex including dATP was obtained. The dATP did not pair with PdG, but instead with the 5'-neighboring T, utilizing Watson-Crick geometry. Thus, all three ternary complexes were of the "type II" structure described for ternary complexes with native DNA [Ling, H., Boudsocq, F., Woodgate, R., and Yang, W. (2001) Cell 107, 91-102]. The PdG adduct remained in the anti conformation about the glycosyl bond in each of these threee ternary complexes. These results provide insight into how -1 frameshift mutations might be generated for the PdG adduct, a structural model for the exocylic M 1dG adduct formed by malondialdehyde.     

Pea chloroplast DNA primase: characterization and role in initiation of replication

A DNA primase activity was isolated from pea chloroplasts and examined for its role in replication. The DNA primase activity was separated from the majority of the chloroplast RNA polymerase activity by linear salt gradient elution from a DEAE-cellulose column, and the two enzyme activities were separately purified through heparin-Sepharose columns. The primase activity was not inhibited by tagetitoxin, a specific inhibitor of chloroplast RNA polymerase, or by polyclonal antibodies prepared against purified pea chloroplast RNA polymerase, while the RNA polymerase activity was inhibited completely by either tagetitoxin or the polyclonal antibodies. The DNA primase activity was capable of priming DNA replication on single-stranded templates including poly(dT), poly(dC), M13mp19, and M13mp19_+ 2.1, which contains the AT-rich pea chloroplast origin of replication. The RNA polymerase fraction was incapable of supporting incorporation of ³H-TTP in in vitro replication reactions using any of these single-stranded DNA templates. Glycerol gradient analysis indicated that the pea chloroplast DNA primase (115–120 kDa) separated from the pea chloroplast DNA polymerase (90 kDa), but is much smaller than chloroplast RNA polymerase. Because of these differences in size, template specificity, sensitivity to inhibitors, and elution characteristics, it is clear that the pea chloroplast DNA primase is an distinct enzyme form RNA polymerase. In vitro replication activity using the DNA primase fraction required all four rNTPs for optimum activity. The chloroplast DNA primase was capable of priming DNA replication activity on any single-stranded M13 template, but shows a strong preference for M13mp19+2.1. Primers synthesized using M13mp19+2.1 are resistant to DNase I, and range in size from 4 to about 60 nucleotides.     

Stimulation by N6,O2'-dibutyrul andenosine 3',5'-cyclic monophosphate of RNA and DNA synthesis and of cell proliferation of rat hepatocytes in primary tissue culture.

The effects of DBcAMP in doses from 1.5 x 10(-8) to 1.5 x 10(-3) M on the compartmental apparent surface area (ASA) and (5(-3H)uridine radioactivity concentration (URC), (methyl-3H)thymidine labelling index per 1 hour ([Me-3H]Tdr LI/h) and per cent mitotic index (MI%) and colchicine metaphase index (CMI%) of young rat differentiated hepatocytes in primary tissue culture were investigated by morphometric and radioautographie methods. In these cells DBcAMP was found to elicit: (1) progressive increments in the ASA of nucleoli, karyoplasm and cytoplasm; (2) peak increases in nucleolar URC at 1.5 x 10(-8) and 10(-5) M, but a slight decrease at 1.5 x 10(-3) M; (3) singificant increments in karyoplasmic and total nuclear URC at all doses, except at 1.5 x 10(-6) and 10(-4) M, when such parameters remained at control levels; (4) steady and progressive increases in cytoplasmic and total cell URC values; (5) marked increments in (Me-3H)Tdr LI/h, MI% and CMI% up to the dose of 1.5 x 10(-4) M, but at 1.5 x 10(-3) M these parameters were found to be either much less enhanced or to approach closely to control values. cAMP in doses from 1.5 x 10(-8) to 10(-4) M also markedly incremented the in vitro hepatocyte CMI%, while having a lesser stimulatory effect at 1.5 x 10(-3)M. Finally of the various possible metabolites of DBcAMP administered at 1.5 x 10(-8) M to liver cultures, N6- and O2'-MBcAMP and, again, cAMP significantly increased the CMI%, of cultured hepatocytes, whereas 5'-AMP, adenosine and allantoin had no significant effect and Na-butyrate slightly decreased it. The present observations strengthen the hypothesis that cAMP and its butyrated derivatives, by possibly amplifying the template activity of the liver chromatin, accelerate the flow of differentiated primary young rat hepatocytes into the various stages of the mitotic cell cycle.     

Accessibility to topoisomerases I and II regulates the replication efficiency of simian virus 40 minichromosomes.

We determined the effects of chromatin structure on template accessibility to replication factors and used three different templates as substrates for simian virus 40 (SV40) DNA replication in vitro: native and salt-treated SV40 minichromosomes and protein-free SV40 DNA. Native minichromosomes contain histone H1 and numerous nonhistone proteins in addition to the core histones, whereas salt-treated minichromosomes carry essentially only core histones. We reasoned that the less densely packed salt-treated minichromosomes should be more effective replication templates due to their more extended configuration. However, contrary to this expectation, we found that native minichromosomes replicated with significantly higher efficiency than salt-treated minichromosomes, while protein-free DNA was most active as a replication template. The higher replication efficiency of native minichromosomes was due to two activities bound to the chromatin, which were identified as DNA topoisomerases I and II. By using chromatin substrates of different general configurations, we also showed that the overall chromatin structure determines accessibility to topoisomerases I and II and thereby the efficiency of replicative chain elongation.     

Boric acid and salinity effects on maize roots. Response of aquaporins ZmPIP1 and ZmPIP2, and plasma membrane H+-ATPase, in relation to water and nutrient uptake

Under saline conditions, an optimal cell water balance, possibly mediated by aquaporins, is important to maintain the whole-plant water status. Furthermore, excessive accumulation of boric acid in the soil solution can be observed in saline soils. In this work, the interaction between salinity and excess boron with respect to the root hydraulic conductance (L₀), abundance of aquaporins (ZmPIP1 and ZmPIP2), ATPase activity and root sap nutrient content, in the highly boron- and salt-tolerant Zea mays L. cv. amylacea, was evaluated. A downregulation of root ZmPIP1 and ZmPIP2 aquaporin contents were observed in NaCl-treated plants in agreement with the L₀ measurements. However, in the H₃BO₃-treated plants differences in the ZmPIP1 and ZmPIP2 abundance were observed. The ATPase activity was related directly to the amount of ATPase protein and Na⁺ concentration in the roots, for which an increase in NaCl- and H₃BO₃+ NaCl-treated plants was observed with respect to untreated and H₃BO₃-treated plants. Although nutrient imbalance may result from the effect of salinity or H₃BO₃ alone, an ameliorative effect was observed when both treatments were applied together. In conclusion, our results suggest that under salt stress, the activity of specific membrane components can be influenced directly by boric acid, regulating the functions of certain aquaporin isoforms and ATPase as possible components of the salinity tolerance mechanism.