Purification and characterization of varicella-zoster virus-induced DNA polymerase.

Infection of WI-38 human fibroblasts with varicella-zoster virus led to the stimulation of host cell DNA polymerase synthesis and induction of a new virus-specific DNA polymerase. This virus-induced DNA polymerase was partially purified and separated from host cell enzymes by DEAE-cellulose and phosphocellulose column chromatographies. This virus-induced enzyme could be distinguished from host cell enzyme by its chromatographic behavior, template specificity, and its requirement of salt for maximal activity. The enzyme could efficiently use poly(dC).oligo(dG)12-18 as well as poly(dA).oligo(dT)12-18 as template-primers. It required Mg2+ for maximal polymerization activity and was sensitive to phosphonoacetic acid, to which host alpha- and beta-DNA polymerase were relatively resistant. In addition, this induced DNA polymerase activity was enhanced by adding 60 mM (NH4)2SO4 to the reaction mixture.     

Human cytomegalovirus. III. Virus-induced DNA polymerase.

Infection of WI-38 human fibroblasts with human cytomegalovirus (CMV) led to the stimulation of host cell DNA polymerase synthesis and induction of a novel virus-specific DNA polymerase. This cytomegalovirus-induced DNA polymerase was purified and separated from host cell enzymes by DEAE-cellulose and phosphocellulose column chromatographies. It can be distinguished from host cell enzymes by chromatographic behavior, template primer specificity, sedimentation property, and the requirement of salt for maximal activity. This virus-induced enzyme has a sedimentation coefficient of 9.2S and is found in both the nuclei and cytoplasm of virus-infected cells, but not in uninfected cells. This enzyme could efficiently use activated calf-thymus DNA, oly(dA)-oligo(dT)12-18, and poly(dC)-oligo(dG)12-18 as template primers, especially poly(dA)-oligo(dT)12-18, but it could not use poly(rA)-oligo(dT)12-18, poly(rC)-oligo(dG)12-18, or oligo(dT)12-18. The enzyme requires Mg2+ for maximal activity, is sensitive to p-hydroxymercuribenzoate, and is not a zinc metalloenzyme. In addition, the cytomegalovirus-induced DNA polymerase activity can be enhanced by adding 0.06 to 0.12 M NaCl or 0.03 to 0.06 M (NH4)2SO4 to the reaction mixture.     

Calf thymus DNA polymerase delta independent of proliferating cell nuclear antigen (PCNA).

DNA polymerase delta from calf thymus was purified under conditions that minimized proteolysis to a specific activity of 27,000 units/mg. The four step isolation procedure included phosphocellulose, hydroxyapatite, heparin-Sepharose and FPLC-MonoS. This enzyme consists of four polypeptides with Mr of 140, 125, 48 and 40 kilodaltons. Velocity gradient sedimentation in glycerol removed the 48 kDa polypeptide while the other three sedimented with the DNA polymerase activity. The biochemical properties of the three subunit enzyme and the copurification of 3'----5' exonuclease activity were typical for a bona fide DNA polymerase delta. Tryptic peptide analysis showed that the 140 kDa polypeptide was different from the catalytic 180 kDa polypeptide of calf thymus DNA polymerase alpha. Both high Mr polypeptides (140 and 125 kDa) were catalytically active as analysed in an activity gel. Four templates were used by DNA polymerase delta with different preferences, namely poly(dA)/oligo(dT)12-18 much much greater than activated DNA greater than poly(dA-dT) greater than primed single-stranded M13DNA. Calf thymus proliferating cell nuclear antigen (PCNA) could not stimulated this DNA polymerase delta in any step of the isolation procedure. If tested on poly(dA)/oligo(dT)12-18 (base ratio 10:1), PCNA had no stimulatory effect on DNA polymerase delta when tested with low enzyme DNA ratio nor did it change the kinetic behaviour of the enzyme. DNA polymerase delta itself did not contain PCNA. The enzyme had an intrinsic processivity of several thousand bases, when tested either on the homopolymer poly(dA)/oligo(dT)12-18 (base ratio 64:1) or on primed single-stranded M13DNA. Contrary to DNA polymerase alpha, no pausing sites were seen with DNA polymerase delta. Under optimal in vitro replication conditions the enzyme could convert primed single-stranded circular M13 DNA of 7,200 bases to its double-stranded form in less than 10 min. This supports that a PCNA independent DNA polymerase delta exists in calf thymus in addition to a PCNA dependent enzyme (Lee, M.Y.W.T. et al. (1984) Biochemistry 23, 1906-1913).     

Biochemical and functional comparison of DNA polymerases alpha, delta, and epsilon from calf thymus.

DNA polymerase alpha, delta and epsilon can be isolated simultaneously from calf thymus. DNA polymerase delta was purified to apparent homogeneity by a four-column procedure including DEAE-Sephacel, phenyl-Sepharose, phosphocellulose, and hydroxylapatite, yielding two polypeptides of 125 and 48 kDa, respectively. On hydroxylapatite DNA polymerase delta can completely be separated from DNA polymerase epsilon. By KCl DNA polymerase delta is eluted first, while addition of potassium phosphate elutes DNA polymerase epsilon. DNA polymerases delta and epsilon could be distinguished from DNA polymerase alpha by their (i) resistance to the monoclonal antibody SJK 132-20, (ii) relative resistance to N2-[p-(n-butyl)phenyl]-2-deoxyguanosine triphosphate and 2-[p-(n-butyl)anilino]-2-deoxyadenosine triphosphate, (iii) presence of a 3'----5' exonuclease, (iv) polypeptide composition, (v) template requirements, (vi) processivities on the homopolymer poly(dA)/oligo(dT12-18), and (vii) lack of primase. The following differences of DNA polymerase delta to DNA polymerase epsilon were evident: (i) the independence of DNA polymerase epsilon to proliferating cell nuclear antigen for processivity, (ii) utilization of deoxy- and ribonucleotide primers, (iii) template requirements in the absence of proliferating cell nuclear antigen, (iv) mode of elution from hydroxylapatite, and (v) sensitivity to d2TTP and to dimethyl sulfoxide. Both enzymes contain a 3'----5' exonuclease, but are devoid of endonuclease, RNase H, DNA helicase, DNA dependent ATPase, DNA primase, and poly(ADP-ribose) polymerase. DNA polymerase delta is 100-150 fold dependent on proliferating cell nuclear antigen for activity and processivity on poly(dA)/oligo(dT12-18) at base ratios between 1:1 to 100:1. The activity of DNA polymerase delta requires an acidic pH of 6.5 and is also found on poly(dT)/oligo(dA12-18) and on poly(dT)/oligo(A12-18) but not on 10 other templates tested. All three DNA polymerases can be classified according to the revised nomenclature for eukaryotic DNA polymerases (Burgers, P.M. J., Bambara, R. A., Campbell, J. L., Chang, L. M. S., Downey, K. M., Hübscher, U., Lee, M. Y. W. T., Linn, S. M., So, A. G., and Spadari, S. (1990) Eur. J. Biochem. 191, 617-618).     

Unique requirements for template primers of DNA polymerase beta from rat ascites hepatoma AH130 cells.

The optimal condition for the rat DNA polymerase beta activity with (rA)n . (dT)12-18 as a template-primer was determined. The activity was remarkably affected by the concentration of the primer, (dT)12-18' and the mixing ratio of (dT)12-18 to (rA)n. DNA polymerase beta requires higher primer concentration (Km = 11.1 microM with respect to 3'-OH of the primer) than DNA polymerase gamma (Km = 0.04 microM) or oncornaviral DNA polymerase (Km = 0.08 microM) and the enzyme represented the maximum activity in the base ratio of 2:1 with (dT)12-18 and (rA)n suggesting the difference in reaction mechanisms of these enzymes. Under the optimized conditions, the specific activity of the near homogeneous preparation of DNA polymerase beta was 1,000,000 units per mg protein.     

RNA-dependent DNA polymerase from a cell line derived from the bone marrow of a patient with polycythemia vera.

A RNA-dependent DNA polymerase was isolated from a human cell line derived from the bone marrow of a patient with polycythemia vera. The purification procedure included chromatography on phosphocellulose and oligo(dT)-cellulose, and glycerol gradient centrifugation. The enzyme could be distinguished from polymerase A by salt elution from phosphocellulose, utilization of poly(rC) - oligo(dG) and its molecular size of about 70000, as determined by centrifugation. Throughout the purification procedure ribonuclease H activity was co-purified. Upon dodecylsulfate-polyacrylamide electrophoresis on microgradient gels two main bands with molecular weights of 68000 and 66000 and three minor bands were detected. The enzyme preferentially used poly(rA) - oligo(dT) as template-primer compared with poly(dA) - oligo(dT). It incorporated dGMP into polymer on poly(rC) - oligo(dG).     

鼻咽癌转移淋巴结中EB病毒相关的DNA多聚酶的分离及其特性研究

一种新的DNA多聚酶已从鼻咽癌(NPC)转移淋巴结胞核酶液,通过DEAE-纤维素柱层析而被部分纯化,并可与细胞的α-及β-DNA多聚酶分开。 此酶有下列特点可与细胞DNA多聚酶区分:(1)可放DEAE-纤维素吸附,需用130mMK_2HPO_4缓冲液方可洗脱下来。(2)可被浓盐所激活,150——200mMKCl或75mM(NH_4)_2SO_4可使它显示最高的酶活性。(3)最适pH为8.0。(4)对磷酰甲酸盐的抑制较敏感。(5)能很好地利用某些合成模板,如poly(dA)·oligo(dT)_(10)及poly(dA)·oligo(dT)_(12-18)。但不能利用poly(rA)·oligo(dT)_(10),证明此酶并非细胞的γ-DNA多聚酶,而与巴基特淋巴瘤的EB病毒相关的(EBV)DNA多聚酶性质十分相似。对照的Raji细胞未见此种EBV-DNA多聚酶。 从鼻咽癌淋巴结中分离出此种EBV-DNA多聚酶,将对EB病毒与NPC的发病关系提供新的证据。     

Identification of oligothymidylates as new simple substrates for Escherichia coli DNA photolyase and their use in a rapid spectrophotometric enzyme assay

Escherichia coli DNA photolyase exhibits the same turnover number (3.4 min-1) for the repair of dimers in oligothymidylates [oligo(dT)n] containing 4-18 thymine residues. This rate is identical with that observed with polythymidylate and with native DNA. The enzyme exhibits a similar high affinity with oligomers containing seven or more thymine residues. A decrease in affinity is detectable with oligo(dT)n when n = 4-6. The enzyme is active with oligo(dT)3, but no evidence for saturation was obtained at dimer concentrations up to 15 microM where the observed repair rate is 43% of the turnover number observed with the higher homologues. Nearly quantitative (90-100%) repair is observed with oligo(dT)n when n is greater than or equal to 9. Photolyase can repair internal dimers and dimers at a 5' end where the terminal ribose is phosphorylated but not at unphosphorylated 5' or 3' ends. The latter can explain a progressive decrease in the extent of repair observed with short-chain oligomers. The observed specificity can also explain why the enzyme is inactive with oligo(dT)2 [p(dT)2] since the only dimer possible in oligo(dT)2 involves an unphosphorylated 3' end. That the enzyme can repair dimers in short-chain, single-stranded analogues for DNA suggests that in catalysis with DNA recognition of the dimer itself is important as opposed to recognition of the deformation in DNA structure produced by the dimer. Dimer repair with oligo(dT)n is detected by the increase in absorbance at 260 nm, a feature which is used as the basis for a rapid spectrophotometric assay with a lower detection limit around 150 pmol of dimer repaired.     

DNA Polymerase of Murine Sarcoma-Leukemia Virus: Lack of Detectable RNase H and Low Activity With Viral RNA and Natural DNA Templates

Kirsten murine sarcoma-leukemia virus (Ki-MSV[MLV]) was found to contain less RNase H per unit of viral DNA polymerase than avian Rous sarcoma virus (RSV). Upon purification by chromatography on Sephadex G-200 and subsequent glycerol gradient sedimentation the avian DNA polymerase was obtained in association with a constant amount of RNase H. By contrast, equally purified DNA polymerase of Ki-MSV(MLV) and Moloney [Mo-MSV(MLV)] lacked detectable RNase H if assayed with two homopolymer and phage fd DNA-RNA hybrids as substrates. On the basis of picomoles of nucleotides turned over, the ratio of RNase H to purified avian DNA polymerase was 1:20 and that of RNase H to purified murine DNA polymerase ranged between <1:2,800 and 5,000. Based on the same activity with poly (A).oligo(dT) the activity of the murine DNA polymerase was 6 to 60 times lower than that of the avian enzyme with denatured salmon DNA template or with avian or murine viral RNA templates assayed under various conditions (native, heat-dissociated, with or without oligo(dT) and oligo(dC) and at different template enzyme ratios). The template activities of Ki-MSV(MLV) RNA and RSV RNA were enhanced uniformly by oligo(dT) but oligo(dC) was much less efficient in enhancing the activity of MSV(MLV) RNA than that of RSV RNA. It was concluded that the purified DNA polymerase of Ki-MSV(MLV) differs from that of Rous sarcoma virus in its lack of detectable RNase H and in its low capacity to transcribe viral RNA and denatured salmon DNA. Some aspects of these results are discussed.     

Studies on the activator 1 protein complex, an accessory factor for proliferating cell nuclear antigen-dependent DNA polymerase delta.

Activator 1 (A1) is a multiprotein complex which is essential for proliferating cell nuclear antigen (PCNA)-dependent DNA polymerase delta (pol delta) activity and efficient in vitro DNA synthesis in the SV40 dipolymerase replication system. In this report, we describe the isolation of A1 from HeLa cytosolic extracts. A1 stimulated pol delta activity in singly primed phi X174 DNA or (dA)4500.oligo(dT)12-18 in reactions containing PCNA, single-stranded DNA binding protein (SSB), and ATP. Using this assay, A1 has been extensively purified. Purified preparations contained five discrete subunits of 145, 40, 38, 37, and 36.5 kDa. ATP hydrolysis to ADP and Pi is essential for A1-dependent pol delta activity, and we have shown that A1 contains an intrinsic ATPase which is stimulated by DNA. The DNA-dependent hydrolysis of ATP can be stimulated by PCNA and further activated by PCNA plus the human single-stranded DNA binding protein. These stimulatory effects were observed with (dA)4500.oligo(dT)12-18, but were not detected with each poly-deoxynucleotide alone. Furthermore, A1 formed a complex with (dA)4500.oligo(dT)12-18 which could be measured by nitrocellulose binding. No complex with (dA)4500 or oligo(dT)12-18 alone was detected by this procedure. Data are also presented which indicate that A1, in conjunction with PCNA, functions as a primer-recognition factor for pol delta, increasing its ability to utilize low levels of primer ends, but it does not increase the size of the DNA products. A1 also markedly reduced the amount of PCNA required for pol delta activity on a multiply primed DNA suggesting that PCNA interacts with A1 at the primer end. These multiple effects of A1 closely resemble the properties of the multisubunit protein RF-C described by Tsurimoto and Stillman (Tsurimoto, T., and Stillman, B. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1023-1027).     

Characterization of an alpha-like DNA polymerase from Bombyx mori silkglands.

A soluble DNA polymerase has been purified near to homogeneity from Bombyx mori silkglands. The following characteristics were observed: high molecular weight (about 150 000 - 220 00); optimum pH about 8; inhibition by high salt concentrations, sulfhydryl-group blocking agents and polyamines; absence of nuclease activity; preference for magnesium as required divalent cation with all the efficient template-primers tested; and clear template-primer specificity, the purified enzyme being able to copy primed - polydeoxyribonucleotide templates [activated DNA, poly(dA).oligo(dT), poly(dA).oligo(rU)] but not polyribonucleotide chains [poly(rA).oligo(dT), poly(rA).oligo(rU)] in the presence of either Mg++ or MN++. Believed to represent the bulk of silkgland DNA polymerase activity, the purified soluble enzyme most resembles vertebrate DNA polymerases alpha when it is compared to other eukaryotic DNA polymerases as yet characterized.     

Particulate DNA polymerase from the cytoplasm of Xenopus laevis oocytes

A DNA polymerase has been partially purified and characterized from Xenopus laevis stage 6 oocytes. The enzyme is present only in the cytoplasm and has been shown to be able to copy Poly(A) x oligo(dT), to be sensitive to N-ethylmaleimide, and to sediment faster than 4 S in high salt glycerol gradient. The enzyme can be extracted from particulate material which has a density in sucrose gradient ranging from 1.200 to 1.225 g/cc. This particulate material is identified by its ability to use Poly(A) x oligo(dT) as template in an exogenous DNA polymerase reaction and by its endogenous DNA synthesizing capacity.     

Identity of DNA polymerase gamma from synaptosomal mitochondria and rat-brain nuclei

DNA polymerase gamma and mitochondrial DNA polymerase were isolated from brain nuclei and synaptosomes respectively. The presence of a single DNA polymerase in synaptosomal mitochondria was established by chromatography on DEAE-cellulose, phosphocellulose and DNA-cellulose, as well as by sedimentation analysis and isoelectric focusing. A great similarity between the purified nuclear DNA polymerase gamma and the mitochondrial enzyme was found by the following criteria: chromatographic behaviour in three column systems; essentially complete inhibition by N-ethyl-maleimide (2 mM); optimal requirements of Mn2+ (0.1 mM), Mg2+ (5 mM) and pH (8.0); template preferences, poly(A) - (dT)20-25 larger than activated DNA larger than poly(dA) - (dT)12-18; lack of activity on single-stranded polynucleotides and (dT)12-primed mRNA; molecular weight (180000), sedimentation (9.2 S) and isoelectric point (pI 5.4). We therefore conclude that brain nuclear DNA polymerase gamma and synaptosomal mitochondrial DNA polymerase are closely related and may even be identical.     

Processiveness of DNA polymerases. A comparative study using a simple procedure.

In this communication, we describe a simple procedure for analyzing the processiveness of DNA polymerases in general. By choosing conditions for which the number of incorporations per available primer is less than 1, we have reduced the probability of a primer molecule being utilized by the enzyme more than once. The primer-template used was poly(dA)300:oligo(dT)10, and the product was isolated by oligo(dT)-cellulose chromatography. The number of dTMP residues added per association was determined from the [3H]dThd + [3'-3H]dTMP/[3H]dThd ratio of the product after its digestion by micrococcal nuclease and spleen phosphodiesterase. Using this procedure, we have found that Escherichia coli DNA polymerase I, T4 DNA polymerase, and calf thymus alpha- and beta-DNA polymerase are "quasi-processive." Most of these enzymes add on the average approximately 10 to 15 nucleotides before dissociating from the template. T5 DNA polymerase, on the other hand, is processive, i.e. it continues to replicate a given template until it is very close to the 5' end of the template. With "nicked DNA-like" poly(dA):oligo(dT), the processiveness of E. coli DNA polymerase I is increased 2- to 2.5-fold. The significance of this increase in determining the "patch size" during DNA repair is discussed.     

Binding properties of avian myeloblastosis virus DNA polymerases to nucleic acid affinity columns.

A new method for the analysis and purification of the RNA-directed DNA polymerase of RNA tumor viruses has been developed. This nucleic acid affinity chromatography system utilizes an immobilized oligo (dT) moiety annealed with poly (A). The alpha and alphabeta DNA polymerases of avain myeloblastosis virus bound effectively to poly (A) oligo (dT)-cellulose. Alpha DNA polymerase did not bind effectively to poly (A) oligo (dT)-cellulose, poly (A)-cellulose, or to cellulose. Alphabeta bound to oligo (dT)-cellulose and cellulose at the same extent (approximately 30%), indicating that this enzyme did not bind specifically to the oligo (DT) moiety only. However, alphabeta bound to poly (A)-cellulose two to three times better than to cellulose itself, showing that alphabeta could bind to poly (A) without a primer. Alphabeta DNA polymerase also bound to poly (C)-cellulose, whereas alpha did not. These data show that the alpha DNA polymerase is defective in binding to nucleic acids if the beta subunit is not present. Data is presented which demonstrates that the alphabeta DNA polymerase bound tighter to poly (A). oligo (DT)-cellulose and to calf thymus DNA-cellulose than the alpha DNA polymerase, suggesting that the beta subunit or, at least part of it is responsible for this tighter binding. In addition, alphabeta DNA polymerase is able to reversibly transcribe avian myeloblastosis virus 70S RNA approximately fivefold faster than alpha DNA polymerase in the presence of Mg2+ and equally efficient in the presence of Mn2+. alpha DNA polymerase transcribed 9S globin m RNA slightly better than alphabeta with either metal ion.     

Novel properties of DNA polymerase beta with poly(rA).oligo(dT) template-primer.

Purified DNA polymerase beta of calf thymus can utilize poly(rA).oligo(dT) as efficiently as poly(dA).oligo(dT) or activated DNA as a template primer. The poly(rA).oligo(dT)-dependent activity of DNA polymerase beta was found to differ markedly from the DNA-dependent activity of the same enzyme (with either activated calf thymus DNA or poly(dA).(dT)10) in the following respects. 1) Poly(rA)-dependent activity was strongly inhibited by natural DNA from various sources or synthetic deoxypolymer duplexes at very low concentrations (less than 0.5 microgram/ml) at which the DNA-dependent activity was affected to a much smaller extent, if at all. 2) Poly(rA)-dependent activity was inhibited by N-ethylmaleimide more strongly than DNA-dependent activity measured at 37 degrees C, while it was resistant to this reagent at 26 degrees C. 3) The curves of the activity versus substrate concentration were sigmoidal in the poly(rA)-dependent reaction but hyperbolic in the activated DNA-dependent reaction. A kinetic study suggested that the association of beta-enzyme protomers may be required to copy the poly(rA) strand.