Characterization Of DNA Polymerase Research Articles

Characterization of DNA polymerase δ from deep-sea hydrothermal vent shrimp Rimicaris exoculata

Characterization of DNA polymerase δ from deep-sea hydrothermal vent shrimp Rimicaris exoculata

Expression and functional study of VpV262 Pol, a moderately halophilic DNA polymerase from the Vibrio parahaemolyticus phage VpV262

Halophilic organisms are found widely in environments where the salt concentration is higher than 0.2 M. Halophilic proteins isolated from these organisms maintain structural integrity and function under high salt stress, whereas their non-halophilic homologs tend to aggregate and collapse. Here we report for the first time the expression and function of a DNA polymerase (DNAPol) VpV262 Pol, which belongs to DNAPol Family A from Vibrio parahaemolyticus phage VpV262. Enzymatic activity assay revealed that VpV262 Pol possessed 5'-3' polymerase activity as well as 3'-5' proofreading exonuclease activity. VpV262 Pol requires Mg2+ or Mn2+ to catalyze the polymerization reaction. Polymerization activity assay under a wide range of salt concentrations showed that VpV262 Pol maintains the highest polymerase activity with 0-0.3 M of NaCl/KCl and 0-0.5 M KAc (potassium acetate) /KGlc (potassium gluconate) when treated with 0-1 M corresponding salts, in contrast to significantly decreased activity of Phi29 Pol and Taq Pol above 0.2 M. Consistent with typical features of other halophilic proteins, negatively-charged amino acids are more frequently distributed on the surface of VpV262 Pol, contributing to highly solubility and enhanced halotolerance. While 3D-Structure of VpV262 Pol needs to be confirmed by experimental data further, this study here has added a member for the relatively small family of halotolerant DNA polymerase, and provides a valuable reference in isolation and characterization of DNA polymerases from halophilic organisms.

Characterization of DNA polymerase β from Danio rerio by overexpression in E. coli using the in vivo/in vitro compatible pIVEX plasmid

BackgroundEukaryotic DNA polymerase β (pol β), the polymerase thought to be responsible for DNA repair synthesis, has been extensively characterized in rats and humans. However, pol β has not been purified or enzymatically characterized from the model fish species Danio rerio (zebrafish). We used the in vitro/in vivo dual expression system plasmid, pIVEX, to express Danio rerio pol β (Danio pol β) for biochemical characterization.ResultsDanio pol β encoded by the in vitro/in vivo-compatible pIVEX plasmid was expressed in E. coli BL21(DE3), BL21(DE3)pLysS, and KRX, and in vitro as a C-terminal His-tagged protein. Danio pol β expressed in vitro was subject to proteolysis; therefore, bacterial overexpression was used to produce the protein for kinetic analyses. KRX cells were preferred because of their reduced propensity for leaky expression of pol β. The cDNA of Danio rerio pol β encodes a protein of 337 amino acids, which is 2-3 amino acids longer than other pol β proteins, and contains a P63D amino acid substitution, unlike mammalian pol βs. This substitution lies in a hairpin sequence within an 8-kDa domain, likely to be important in DNA binding. We performed extensive biochemical characterization of Danio pol β in comparison with rat pol β, which revealed its sensitivity to metal ion activators (Mn2+ and Mg2+), its optimum salt concentration (10 mM KCl and 50 mM NaCl), alkaline pH optimum (pH 9.0), and low temperature optimum (30°C). Substituting Mn2+ for Mg2+ resulted in 8.6-fold higher catalytic efficiency (kcat/Km).ConclusionsOur characterization of pol β from a model fish organism contributes to the study of the function and evolution of DNA polymerases, which are emerging as important cellular targets for chemical intervention in the development of anticancer agents.

Characterization of DNA polymerase β splicing variants in gastric cancer: The most frequent exon 2-deleted isoform is a non-coding RNA

DNA repair polymerase β (Pol β) gene variants are frequently associated with tumor tissues. In this study a search for Pol β mutants and splice variants was conducted in matched normal and tumor gastric tissues and blood samples from healthy donors. No tumor associated mutations were found while a variety of alternative Pol β splicing variants were detected with high frequency in all the specimens analysed. Quantitative PCR of the Pol β variant lacking exon 2 (Ex2Δ) and the isoforms with exon 11 skipping allowed to clarify that these variants are not tumor- neither tissue-specific and their levels vary greatly among different individuals. The most frequent Ex2Δ variant was further characterized. We clearly demonstrated that this variant does not encode protein, as detected by both western blotting and immunofluorescence analysis of human AGS cells expressing HA-tagged Ex2Δ. The lack of translation was confirmed by comparing the DNA gap-filling capacity and alkylation sensitivity of wild type and Pol β null murine fibroblasts expressing the human Ex2Δ variant. We showed that the Ex2Δ transcript is polyadenylated and its half-life is significantly longer than that of the wild type mRNA as inferred by treating AGS cells with actinomycin D. Moreover, we found that it localizes to polyribosomes suggesting a role as post-transcriptional regulator. This study identifies a new type of DNA repair variants that do not give rise to functional proteins but to non-coding RNAs that could either modulate target mRNAs or represent unproductive splicing events.

Characterization of DNA polymerase from the hyperthermophilic archaeon Thermococcus marinus and its application to PCR

The family B DNA polymerase gene from the archaeon Thermococcus marinus (Tma) contains a long open reading frame of 3,939 bp that encodes 1,312 amino acid residues. The gene is split by one intervening sequence that forms a continuous open reading frame with the two polymerase exteins. In this study, the Tma DNA polymerase gene both with (precursor form) and without (mature form) its intein was expressed in Escherichia coli, purified by heat treatment and HiTrap Heparin HP column chromatography and characterized. Primary sequence analysis of the mature Tma polymerase showed high sequence identity with DNA polymerases in the genus Thermococcus. The expressed precursor form was easily spliced during purification steps. The molecular mass of the purified Tma DNA polymerases is about 90 kDa, as estimated by SDS-PAGE. Both Tma DNA polymerases showed the same properties. PCR performed with this enzyme was found to be optimal in the presence of 50 mM Tris-HCl (pH 8.4), 40 mM KCl, 12.5 mM (NH(4))(2)SO(4,) 2 mM MgCl(2,) 0.05% Triton X-100 and 0.0075% BSA. Furthermore, long-range PCR and time-saving PCR were performed using various specific ratios of Taq and Tma DNA polymerases (Tma plus DNA polymerase).

Characterization of DNA polymerase X from Thermus thermophilus HB8 reveals the POLXc and PHP domains are both required for 3′–5′ exonuclease activity

The X-family DNA polymerases (PolXs) comprise a highly conserved DNA polymerase family found in all kingdoms. Mammalian PolXs are known to be involved in several DNA-processing pathways including repair, but the cellular functions of bacterial PolXs are less known. Many bacterial PolXs have a polymerase and histidinol phosphatase (PHP) domain at their C-termini in addition to a PolX core (POLXc) domain, and possess 3′–5′ exonuclease activity. Although both domains are highly conserved in bacteria, their molecular functions, especially for a PHP domain, are unknown. We found Thermus thermophilus HB8 PolX (ttPolX) has Mg2+/Mn2+-dependent DNA/RNA polymerase, Mn2+-dependent 3′–5′ exonuclease and DNA-binding activities. We identified the domains of ttPolX by limited proteolysis and characterized their biochemical activities. The POLXc domain was responsible for the polymerase and DNA-binding activities but exonuclease activity was not detected for either domain. However, the POLXc and PHP domains interacted with each other and a mixture of the two domains had Mn2+-dependent 3′–5′ exonuclease activity. Moreover, site-directed mutagenesis revealed catalytically important residues in the PHP domain for the 3′–5′ exonuclease activity. Our findings provide a molecular insight into the functional domain organization of bacterial PolXs, especially the requirement of the PHP domain for 3′–5′ exonuclease activity.

Gene structure, purification and characterization of DNA polymerase β from Xiphophorus maculatus

Cloning of the Xiphophorus maculatus Polβ gene and overexpression of the recombinant Polβ protein has been performed. The organization of the XiphPolβ introns and exons, including intron–exon boundaries, have been assigned and were found to be similar to that for human Polβ with identical exon sizes except for exon XII coding for an additional two amino acid residues in Xiphophorus. The cDNA sequence encoding the 337-amino acid X. maculatus DNA polymerase β (Polβ) protein was subcloned into the Escherichia coli expression plasmid pET. Induction of transformed E. coli cells resulted in the high-level expression of soluble recombinant Polβ, which catalyzed DNA synthesis on template–primer substrates. The steady-state Michaelis constants (Km) and catalytic efficiencies (kcat/Km) of the recombinant XiphPolβ for nucleotide insertion opposite single-nucleotide gap DNA substrates were measured and compared with previously published values for recombinant human Polβ. Steady-state in vitro Km and kcat/Km values for correct nucleotide insertion by XiphPolβ and human Polβ were similar, although the recombinant Xiphophorus protein exhibited 2.5–7-fold higher catalytic efficiencies for dGTP and dCTP insertion versus human Polβ. In contrast, the recombinant XiphPolβ displayed significantly lower fidelities than human Polβ for dNTP insertion opposite a single-nucleotide gap at 37 °C.

Characterization of DNA polymerase δ from a higher plant, rice ( Oryzasativa L.)

DNA polymerase δ (pol δ), which is comprised of at least two essential subunits, is an important enzyme involved in DNA replication and repair. We have cloned and characterized both the catalytic and small subunits of pol δ from rice ( Oryza sativa L. cv. Nipponbare). The open reading frames of OsPolδ1 and δ2 encoded a predicted product of 1105 amino acid residues with a molecular weight of 124 kDa for OsPolδ1, and of 429 residues with a molecular weight of 48 kDa for OsPolδ2. Northern blotting analysis indicated that OsPolδ1 and δ2 transcripts were expressed strongly in proliferating tissues such as shoot apical meristem. The expression patterns of both subunits in the organs were slightly different. Therefore, we analyzed the spatial distribution pattern of OsPolδ1 transcripts by in situ hybridization. In the shoot apex, OsPolδ1 mRNA was abundant in the shoot apical meristem. In the roots, the OsPolδ1 transcript accumulated at high levels in the root apical meristem. In mature leaves, OsPolδ1 was induced after UV irradiation, but OsPolδ2 was not. The amounts of the OsPolδ1 and δ2 mRNAs in the rice cells changed rapidly during cell proliferation. These results indicated that the levels of OsPolδ expression are markedly correlated with cell proliferation, and that some of OsPolδ might have special roles in the leaves.

Cloning, expression, and characterization of DNA polymerase I from the hyperthermophilic archaea Thermococcus fumicolans.

The DNA polymerase I gene of a newly described deep-sea hydrothermal vent Archaea species, Thermococcus fumicolans, from IFREMERS's collection of hyperthermophiles has been cloned in Escherichia coli. As in Thermococcus litoralis, the gene is split by two intervening sequences (IVS) encoding inteins inserted in sites A and C of family B DNA polymerases. The entire DNA polymerase gene, containing both inteins, was expressed at 30 degrees C in E. coli strain BL21(DE3)pLysS using the pARHS2 expression vector. The native polypeptide precursor of 170kDa was obtained, and intein splicing as well as ligation of the three exteins was observed in vitro after heat exposure. The recombinant enzyme was purified and some of its activities were characterized: polymerization, thermostability, exonuclease activities, and fidelity.

Cloning, purification and characterization of DNA polymerase beta from Xenopus laevis--studies on its potential role in DNA-end joining.

Double-strand breaks in the DNA of vertebrate cells are joined by mechanisms of non-homologous DNA-end joining (NEJ). In extracts from Xenopus eggs, NEJ is inhibited by dideoxynucleotides, indicating a possible involvement of DNA polymerase beta (Pol beta). Since some types of NEJ products were shown to be formed in vitro by prokaryotic DNA polymerases lacking exonuclease activity, we were interested in whether Pol beta alone would be capable of catalyzing NEJ reactions. Therefore we have cloned the full-length cDNA of the Xenopus laevis Pol beta. The cDNA, predicting a highly conserved 39-kDa protein of 334 amino acids, was tagged with six histidine residues at its N-terminus for overexpression in Escherichia coli, purified to near homogeneity, and shown to have the same catalytic properties as the previously cloned rat and human enzymes. Using oligonucleotides as substrates we show that the recombinant Xenopus Pol beta adds single untemplated nucleotides to blunt ends. However, under conditions that permit efficient NEJ in Xenopus egg extracts, Pol beta does not form those types of NEJ products formed by the prokaryotic polymerases indicating that Pol beta alone is not able to mediate the complex NEJ process in vitro. Using substrates with 3' protruding single strands of increasing length (6-16 nucleotides) we show that Pol beta initiates fill-in DNA synthesis on fold-back structures formed by the longest 3' protruding stand. This unusual feature of beta-type polymerases requires that the loop of the fold-back structure consists of at least six bases and the stem be paired by at least 2 bp to facilitate priming of DNA synthesis.

Characterization of DNA polymerase from Pyrococcus sp. strain KOD1 and its application to PCR.

The DNA polymerase gene from the archaeon Pyrococcus sp. strain KOD1 (KOD DNA polymerase) contains a long open reading frame of 5,013 bases that encodes 1,671 amino acid residues (GenBank accession no. D29671). Similarity analysis revealed that the DNA polymerase contained a putative 3'-5' exonuclease activity and two in-frame intervening sequences of 1,080 bp (360 amino acids; KOD pol intein-1) and 1,611 bp (537 amino acids; KOD pol intein-2), which are located in the middle of regions conserved among eukaryotic and archaeal alpha-like DNA polymerases. The mature form of the DNA polymerase gene was expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. 3'-5' exonuclease activity was confirmed, and although KOD DNA polymerase's optimum temperature (75 degrees C) and mutation frequency (3.5 x 10(-3)) were similar to those of a DNA polymerase from Pyrococcus furiosus (Pfu DNA polymerase), the KOD DNA polymerase exhibited an extension rate (100 to 130 nucleotides/s) 5 times higher and a processivity (persistence of sequential nucleotide polymerization) 10 to 15 times higher than those of Pfu DNA polymerase. These characteristics enabled the KOD DNA polymerase to perform a more accurate PCR in a shorter reaction time.

Purification and characterization of DNA polymerases from Plasmodium falciparum

Fractionation of Plasmodium falciparum cellular extracts by fast protein liquid chromatography (FPLC) identified at least two different DNA polymerases. An aphidicolin-sensitive activity co-purified with a primase activity. This, in combination with other characteristics (processivity, sensitivity to other inhibitors), most likely classifies this enzyme as an α-like DNA polymerase. It was, however, relatively resistant to N 2-( p- n-butylphenyl)deoxyguanosine 5′-triphosphate (IC 50 = 6.6 μM) and differs in this aspect from the host homologue, possibly indicating structural differences between host and parasite DNA polymerase α. The other DNA polymerase matched eukaryotic DNA polymerase γ in all properties tested.

Characterization of dna polymerase α from untransformed and pSV3.neo-transformed human fibroblasts

1. 1. The specific activity of DNA-polymerase α isolated from pSV3.neo-transformed cells was more than 9-fold higher than that of polymerase a from untransformed cells. 2. 2. Western blot analysis, using anti-SV40 large T antigen, of both a crude cellular extract and of partially purified polymerase a from pSV3.neo-transformed cells revealed a single 76 kDa immunoreactive band not found in either crude extracts or partially purified enzyme from untransformed cells. 3. 3. The α polymerases from untransformed and transformed cells differed in molecular size, sensitivity to various inhibitors, specificity of template-primer utilization, and binding affinity for DNA cellulose, but showed essentially no differences in K m or V max . 4. 4. These data suggest that polymerase α isolated from pSV3.neo-transformed cells exhibits altered physical and catalytic characteristics compared with its untransformed cell counterpart, and that those alterations may be associated with increased replication of the genome in plasmid-transformed cells.

Purification and characterization of DNA polymerases from Plasmodium berghei

DNA polymerases from the malaria parasite Plasmodium berghei were purified more than 50-fold. Several distinct enzymatic activities were isolated that could be distinguished by the use of various specific DNA polymerase inhibitors. In particular, subdivision into an aphidicolin-sensitive and an aphidicolin-resistant group was possible. Further analysis allowed a better comparison with host DNA polymerases and indicated that one aphidicolin-sensitive DNA polymerase resembled DNA polymerase α displaying processive DNA synthesis and using RNA primers, whereas another aphidicolin-sensitive DNA polymerase was distributive and only used DNA primers. Marked differences from the host enzymes do exist, however, such as insensitivity to BuPdGTP. Another P. berghei DNA polymerase was isolated that showed characteristics of a DNA polymerase β-like enzyme, but which differed from host DNA polymerase β in its insensitivity to dideoxynucleotides.

Characterization of DNA polymerase α activity from a mouse DNA temperature-sensitive mutant, strain tsFT20, which shows a defect in DNA polymerase α activity at restrictive temperatures

tsFT20 cells derived from mouse FM3A cells are DNA temperature-sensitive mutants, which have heat-labile DNA polymerase α activity. When tsFT20 cells were incubated at restrictive temperatures, intracellular levels of DNA polymerase α activity changed biphasically, showing an initial fast decrease (phase I) and a subsequent slow decrease (phase II). The activity of DNA polymerase α from tsFT20 cells cultured at a permissive temperature (33 °C) was greatly increased by the addition of glycerol or ethylene glycol to the reaction mixture, while little increase in enzyme activity was observed at any concentration of glycerol or ethylene glycol tested with the enzyme from the cells cultured at a restrictive temperature (39 °C) for 8 h (phase II). The activity of DNA polymerase α from wild-type cells was also increased by the addition of glycerol but the increase was much less than that in the tsFT20 cells. An in vitro preincubation experiment showed that DNA polymerase α from tsFT20 cells cultured at 33 °C very rapidly lost its ability to be stimulated by glycerol. Furthermore, the experiment using the extracts prepared from tsFT20 cells cultured at 39 °C for various periods showed that the ability to be stimulated by glycerol decreased with the duration of incubation time at 39 °C. DNA polymerase α from the revertants, which can grow at 39 °C and exhibit a partial recovery in heat stability of DNA polymerase α activity, showed an intermediate response to glycerol, between those of DNA polymerase α from tsFT20 and from the wild-type cells. Finally, it was observed that the level of enzyme activity that can be stimulated by glycerol correlated well with the DNA synthesizing ability of tsFT20 Cells.

Characterization of DNA polymerase β mRNA: cell-cycle and growth response in cultured human cells

DNA polymerase beta (beta-polymerase) is a housekeeping enzyme involved in DNA repair in vertebrate cells. We used a cDNA probe to study abundance of beta-polymerase mRNA in cultured human cells. The mRNA level in synchronized HeLa cells, representing different stages of the cell-cycle, varied only slightly. Contact inhibited fibroblasts AG-1522 contained the same level of mRNA as growing cells. The steady-state level of mRNA in fibroblasts is equivalent to 6 molecules per cell. The results indicate that the beta-polymerase transcript is "low abundance" and is neither cell-cycle nor growth phase responsive.

Purification and characterization of DNA polymerase from the archaebacterium Methanobacterium thermoautotrophicum.

Purification and characterization of DNA polymerase from the archaebacterium Methanobacterium thermoautotrophicum.

Characterization of DNA polymerase I*, a form of DNA polymerase I found in Escherichia coli expressing SOS functions.

DNA polymerase I* is a form of the DNA polymerase I isolated from Escherichia coli which are expressing recA/lexA (SOS) functions. Induction of recA or polA1 cells by nalidixic acid does not result in the appearance of pol I*, but lexA or recA mutants that are constitutive for SOS functions constitutively express pol I* and mutants which lack functional recA protein produce pol I* when they carry a lexA mutation which renders the lexA repressor inoperative. Pol I* has been induced by nalidixic acid in dinA, dinD, dinF, and umuC mutants. Polymerase I* has a lower affinity for single-stranded DNA-agarose than polymerase I and it sediments through sucrose gradients in a dispersed manner between 6.6-10.5 S, whereas polymerase I sediments at 5 S. Whereas pol I* migrates significantly faster than pol I in nondenaturing polyacrylamide gels, the active polypeptide of both forms migrates at the same rate in denaturing polyacrylamide gels. Compared with polymerase I, polymerase I* has an enhanced capacity to incorporate the adenine analog, 2-amino-purine, into activated salmon sperm DNA and a relatively low fidelity in replicating synthetic polydeoxyribonucleotides. Both the 3'----5' (proofreading) and 5'----3' (nick-translational) exonuclease activities of pol I* and pol I are indistinguishable. Estimates of processivity give a value of approximately 6 for both forms of the enzyme.

Isolation and characterization of DNA polymerases from mature oocytes of the starfish, Asterina pectinifera.

Four distinctive DNA polymerases have been isolated from mature oocytes of the starfish, Asterina pectinifera, and characterized. The N-ethylmaleimide-resistant DNA polymerase activity was separated from the other three DNA polymerases by hydroxyapatite column chromatography. The enzyme, whose approximate sedimentation coefficient was 3.3S, effectively utilized poly(dA)-oligo(dT) as a template-primer and was completely inhibited by 10 microM 2', 3'-dideoxythymidine-5'-triphosphate but was not inhibited by aphidicolin. Therefore, this enzyme was classified as a eukaryotic DNA polymerase-beta. Three species of the N-ethylmaleimide-sensitive DNA polymerase were separated with a phosphocellulose column. Two (5.5S and 6.5S) of the enzymes eluted from the column preferred activated DNA as a template-primer. Both of the DNA polymerases were inhibited by aphidicolin but not by 2',3'-dideoxythymidine-5'-triphosphate. Judging from the above results, these two DNA polymerases were classified as eukaryotic DNA polymerase-alpha. The third N-ethylmaleimide-sensitive enzyme (approximately 7S) eluted from the phosphocellulose column utilized poly(rA)-oligo(dT) as well as poly(dA)-oligo(dT) as template-primers. The enzyme activity was resistant to aphidicolin but sensitive to 2',3'-dideoxythymidine-5'-triphosphate. This enzyme resembles eukaryotic DNA polymerase-gamma. Thus, it is concluded that the four species of DNA polymerase found in starfish oocytes resemble eukaryotic DNA polymerase-alpha, -beta, and -gamma.

エールリッヒ腹水癌細胞の核小体に存在するDNAポリメラーゼの諸性質について

エールリッヒ腹水癌細胞の核小体に存在するDNAポリメラーゼの諸性質について