Dideoxynucleoside triphosphate-sensitive DNA polymerase from rice is involved in base excision repair and immunologically similar to mammalian DNA pol beta

A single polypeptide with ddNTP-sensitive DNA polymerase activity was purified to near homogeneity from the shoot tips of rice seedlings and analysis of the preparations by SDS-PAGE followed by silver staining showed a polypeptide of 67 kDa size. The DNA polymerase activity was found to be inhibitory by ddNTP in both in vitro DNA polymerase activity assay and activity gel analysis. Aphidicolin, an inhibitor of other types of DNA polymerases, had no effect on plant enzyme. The 67 kDa rice DNA polymerase was found to be recognized by the polyclonal antibody (purified IgG) made against rat DNA polymerase beta (pol beta) both in solution and also on Western blot. The recognition was found to be very specific as the activity of Klenow enzyme was unaffected by the antibody. The ability of rice nuclear extract to correct G:U mismatch of oligo-duplex was observed when oligo-duplex with 32P-labeled lower strand containing U (at 22nd position) was used as substrate. Differential appearance of bands at 21-mer, 22-mer, and 51-mer position in presence of dCTP was visible only with G:U mismatch oligo-duplex, but not with G:C oligo-duplex. While ddCTP or polyclonal antibody against rat-DNA pol beta inhibits base excision repair (BER), aphidicolin had no effect. These results for the first time clearly demonstrate the ability of rice nuclear extract to run BER and the involvement of ddNTP-sensitive pol beta type DNA polymerase. Immunological similarity of the ddNTP-sensitive DNA polymerase beta of rice and rat and its involvement in BER revealed the conservation of structure and function of ddNTP-sensitive DNA pol beta in plant and animal.     

An insight into the biological functions of family X-DNA polymerase in DNA replication and repair of plant genome

Recently we have reported the characterization of a novel single subunit 62-kDa polypeptide with ddNTP-sensitive DNA polymerase activity from the developing seeds of mungbean (Vigna radiata). The protein showed higher expression and activity level during nuclear endoreduplication stages of mungbean seeds and similarity with mammalian DNA polymerase β in many physicochemical properties.¹ The enzyme was found to specifically interact with PCNA (proliferating cell nuclear antigen),² and expressed in both meristematic and meiotic tissues. Functional assays have demonstrated binding of the enzyme to normal and mismatched DNA substrates and with fidelity DNA synthesis in moderately processive mode, suggesting probable involvement of the enzyme in both replication and recombination.³ Here we have discussed the position of mungbean DNA polymerase as a homologue of DNA Pol λ, one of the newly identified member of family-X DNA polymerase in plants and illustrated the functional relevance of this enzyme in maintaining the coordination between DNA replication and repair in plant genome.Key words: family X-DNA polymerase, DNA polymerase λ, mungbean DNA polymerase, BRCT module, DNA repair     

Enzymatic activities of overexpressed herpes simplex virus DNA polymerase purified from recombinant baculovirus-infected insect cells.

Biochemical characterization of the herpes simplex virus (HSV) DNA polymerase, a model DNA polymerase and an important target for antiviral drugs, has been limited by a lack of pure enzyme in sufficient quantity. To overcome this limitation, the HSV DNA polymerase gene was introduced into the baculovirus, Autographa californica nuclear polyhedrosis virus, under the control of the polyhedrin promoter to give rise to a recombinant baculovirus, BP58. BP58-infected Spodoptera frugiperda insect cells expressed a polypeptide that was indistinguishable from authentic polymerase by several immunological and biochemical properties, at levels approximately ten-fold higher per infected cell than found in HSV-infected Vero cells. The DNA polymerase was purified to apparent homogeneity from BP58-infected insect cells. Using activated DNA as primer-template, the purified enzyme exhibited specific activity similar to that of enzyme isolated from HSV-infected Vero cells, indicating that additional polymerase-associated proteins from HSV-infected cells are not critical for activity with this primer-template. 3'-5' exonuclease activity co-purified with the BP58-expressed HSV DNA polymerase, demonstrating that this activity is intrinsic to the polymerase polypeptide. The purified enzyme also exhibited RNAse H activity. The recombinant baculovirus should permit detailed biochemical and biophysical studies of this enzyme.     

A plant phytotoxin, solanapyrone A, is an inhibitor of DNA polymerase beta and lambda.

Solanapyrone A, a phytotoxin and enzyme inhibitor isolated from a fungus (SUT 01B1-2) selectively inhibits the activities of mammalian DNA polymerase beta and lambda (pol beta and lambda) in vitro. The IC50 values of the compound were 30 microm for pol beta and 37 microm for pol lambda. Because pol beta and lambda are in a family and their three-dimensional structures are thought to be highly similar to each other, we used pol beta to analyze the biochemical relationship with solanapyrone A. On pol beta, solanapyrone A antagonistically competed with both the DNA template and the nucleotide substrate. BIAcore analysis demonstrated that solanapyrone A bound selectively to the N-terminal 8-kDa domain of pol beta. This domain is known to bind single-stranded DNA, provide 5'-phosphate recognition of gapped DNA, and cleave the sugar-phosphate bond 3' to an intact apurinic/apyrimidinic (AP) site (i.e. AP lyase activity) including 5'-deoxyribose phosphate lyase activity. Solanapyrone A inhibited the single-stranded DNA-binding activity but did not influence the activities of the 5'-phosphate recognition in gapped DNA structures and the AP lyase. Based on these results, the inhibitory mechanism of solanapyrone A is discussed.     

Isolation of the DNA polymerase alpha core enzyme from mouse cells

DNA polymerase alpha has been purified from mouse hybridoma cells approximately 30,000-fold using a combination of conventional and high performance liquid chromatography. In contrast to previous characterizations of mammalian DNA polymerase alpha, this enzyme has a single high molecular mass polypeptide (185 kDa) in tight association with a 68-kDa polypeptide and this structure appears to be the core DNA polymerase of the mouse cells. The biochemically purified enzyme, with a specific activity of approximately 200,000 units/mg protein, has an estimated molecular mass by gel filtration chromatography of 240 kDa and sedimentation value of 9 S, consistent with the enzyme being a heterodimer of 185 and 68 kDa. The enzyme is sensitive to both N-ethylmaleimide and aphidicolin and insensitive to ddTTP. Using an activated DNA template, the apparent Km values for the deoxynucleotide triphosphates are approximately 0.5-1 microM. The purified DNA polymerase has neither exonuclease nor primase activities and is the predominant DNA polymerase alpha activity in the mouse cells.     

Partial purification and characterization of a beta-like DNA polymerase from Leishmania mexicana.

Deoxyribonucleic acid polymerase beta (EC 2.7.7.7) from the lower eukaryotic parasitic protozoan Leishmania mexicana has been partially purified over 9,000 fold and characterized for the very first time. Like mammalian DNA polymerase beta the protozoan enzyme is of low molecular weight (40,000), has a broad pH range, and is resistant to inhibition by N-ethylmaleimide and aphidicolin. It is unlike mammalian DNA polymerase beta in utilization of various templates and response to various inhibitors and sensitivity to high ionic strength, but similar to a beta-like enzyme from a related organism Crithidia fasciculata. It is estimated that this enzyme constitutes 20% of the polymerase activity of the crude cell extract.     

Yeast open reading frame YCR14C encodes a DNA beta-polymerase-like enzyme.

We have shown by activity gel that overexpression in E. coli of a yeast chromosome 3 open reading frame (ORF) designated YCR14C and bearing homology to mammalian DNA polymerases beta results in a new DNA polymerase in the host cells. The molecular mass of this enzyme corresponded to the YCR14C-predicted 67 kDa protein, and NH2-terminal amino acid sequencing confirmed that the expressed protein was encoded by the yeast ORF. This new yeast DNA polymerase was purified to homogeneity from E.coli. In a fashion similar to that of mammalian beta-polymerases, the purified yeast enzyme exhibited distributive DNA synthesis on DNA substrate with a single-stranded template and processive gap-filling synthesis on a short-gapped DNA substrate. Activity of this yeast beta-polymerase-like enzyme was sensitive to the beta-polymerase inhibitor ddNTP and resistant to both 1 mM NEM and neutralizing antibody to E. coli DNA polymerase I. These results, therefore, indicate that YCR14C encodes a DNA beta-polymerase-like enzyme in yeast, and we name it DNA polymerase IV. Yeast strains harboring a deletion mutation of the pol IV gene are viable, they exhibit no increase in sensitivity to ultraviolet light, ionizing radiation or alkylating agents, and sporulation and spore viability are not affected in the mutant.     

Tobacco proliferating cell nuclear antigen binds directly and stimulates both activity and processivity of ddNTP-sensitive mungbean DNA polymerase

PCNA is well known as a component of DNA replication system and plays important roles in multiple cellular pathways in addition to replication and repair. In this work we have demonstrated the physical and functional interaction between tobacco PCNA and mungbean ddNTP-sensitive DNA polymerase which shares many physicochemical properties with family X-DNA polymerases except with the moderately processive mode of nucleotide incorporation. We have shown here that recombinant PCNA binds directly to mungbean DNA polymerase as revealed in affinity chromatography, pull-down and co-immunoprecipitation approaches. In vitro DNA polymerase activity assay and processivity analyses indicated recombinant PCNA specifically stimulates both activity and processivity of mungbean DNA polymerase. These observations lead to interesting speculation about the functional significance of the ddNTP-sensitive enzyme in replication event in higher plants since the enzyme has been shown to be active and expressed at an elevated level during the endoreduplication stages in developing mungbean seeds.     

The base substitution fidelity of DNA polymerase beta-dependent single nucleotide base excision repair

Damaged DNA bases are removed from mammalian genomes by base excision repair (BER). Single nucleotide BER requires several enzymatic activities, including DNA polymerase and 5',2'-deoxyribose-5-phosphate lyase. Both activities are intrinsic to four human DNA polymerases whose base substitution error rate during gap-filling DNA synthesis varies by more than 10,000-fold. This suggests that BER fidelity could vary over a wide range in an enzyme dependent manner. To investigate this possibility, here we describe an assay to measure the fidelity of BER reactions reconstituted with purified enzymes. When human uracil DNA glycosylase, AP endonuclease, DNA polymerase beta, and DNA ligase 1 replace uracil opposite template A or G, base substitution error rates are 相似文献   

Isolation and partial characterization of a high-molecular-weight DNA polymerase from Leishmania mexicana.

This paper describes for the first time the isolation and characterization of a high-molecular-weight predominant DNA polymerase from the genus Leishmania, which are parasitic flagellated protozoa. Like mammalian DNA polymerase alpha, the leishmanial DNA polymerase, designated DNA polymerase A, is of high-molecular-weight, is sensitive to N-ethylmaleimide and is inhibited by high ionic strength. Unlike mammalian DNA polymerase alpha, but similar to the predominant DNA polymerase isolated from the related lower eukaryotic organisms, Trypanosoma cruzi and Crithidia fasciculata, the leishmanial DNA polymerase A is resistant to inhibition by aphidicolin, a potent inhibitor of DNA replication in mammalian cells and of DNA polymerase alpha. The DNA polymerase A was purified 28,000-fold and properties such as pH optimum, salt sensitivity, template requirements and response to DNA polymerase inhibitors were determined. A low-molecular-weight DNA polymerase was detected during the isolation procedures, but was separated from the polymerase A activity. Differences in responses to specific antisera and specific mammalian DNA polymerase alpha inhibitors suggest that the leishmanial high-molecular-weight A enzyme is sufficiently different to suggest this enzyme as a chemotherapeutic target.     

Wheat DNA polymerase CI: a homologue of rat DNA polymerase β

We have previously described a low-molecular-weight DNA polymerase (52 kDa) from wheat embryo: DNA polymerase CI (pol CI). This enzyme shares some biochemical properties with animal DNA polymerase (pol ). In this report, we analyse pol CI in wheat embryo germination. Immunodetection and measurement of the enzyme activity show that wheat pol CI remains at a constant level during germination, whereas dramatic changes of the replicative DNA polymerase A and B activities were previously reported. We observe that the level of pol CI in physiological conditions (embryo germination and dividing cell culture) is in agreement with a pol -type DNA polymerase. By microsequencing of the electroblotted 52 kDa polypeptide, we determined the sequence of a dodecapeptide from the N-terminal region. A comparative analysis of the N-terminal pol CI peptide with some mammalian pol sequences shows a clear homology with helix 1 of the N-terminal ssDNA domain (residues 15 to 26) of the rat pol . Thus, the helical structure of this region should be conserved in the wheat peptide. This represents the first evidence of a partial primary structure of a -type DNA polymerase in plants.     

Hydrolysis of 3'-terminal mispairs in vitro by the 3'----5' exonuclease of DNA polymerase delta permits subsequent extension by DNA polymerase alpha

Purified DNA polymerase alpha, the major replicating enzyme found in mammalian cells, lacks an associated 3'----5' proofreading exonuclease that, in bacteria, contributes significantly to the accuracy of DNA replication. Calf thymus DNA polymerase alpha cannot remove mispaired 3'-termini, nor can it extend them efficiently. We designed a biochemical assay to search in cell extracts for a putative proofreading exonuclease that might function in concert with DNA polymerase alpha in vivo but dissociates from it during purification. Using this assay, we purified a 3'----5' exonuclease from calf thymus that preferentially hydrolyzes mispaired 3'-termini, permitting subsequent extension of the correctly paired 3'-terminus by DNA polymerase alpha. This exonuclease copurifies with a DNA polymerase activity that is biochemically distinct from DNA polymerase alpha and exhibits characteristics described for a second replicative DNA polymerase, DNA polymerase delta. In related studies, we showed that the 3'----5' exonuclease of authentic DNA polymerase delta, like the purified exonuclease, removes terminal mispairs, allowing extension by DNA polymerase alpha. These data suggest that a single proofreading exonuclease could be shared by DNA polymerases alpha and delta, functioning at the site of DNA replication in mammalian cells.     

An inhibitor of DNA polymerases alpha and delta in calf thymus DNA.

Most, although not all, samples of commercial calf thymus DNA were strongly inhibitory to DNA polymerase alpha; the inhibition made the DNA useless as a template for this enzyme. In a pre-assembled DNA polymerase assay mixture (minus enzyme but including activated DNA) the inhibition tended to diminish with time but at a rate that was not predictable, and some inhibition usually persisted. It was concluded that the inhibition was the result of contamination of the DNA by a heparin-like material on the basis of the following: 1) the inhibition could be reversed by treatment of the DNA with heparinase; 2) both the endogenous inhibitory effect of calf thymus DNA as well as the inhibitory effect of heparin on DNA polymerase alpha are reversed by protamine (which is known to prevent the antithrombin activity of heparin); 3) both the endogenous inhibition and inhibition by heparin are also reversed by ampholyte (which also prevents the antithrombin activity of heparin); and 4) both the endogenous and the heparin-induced inhibitory effects display the same spectrum of activity against mammalian DNA polymerases, i.e. both DNA polymerases alpha and delta are extremely sensitive whereas, DNA polymerases beta and gamma are resistant. The last result also suggests the use of heparin as a specific inhibitor of purified mammalian DNA polymerases alpha and delta, similar to the use of aphidicolin.     

Characterization of maize polyamine oxidase.

Some structural and biochemical characteristics of polyamine oxidase (PAO) purified from maize shoots have been examined. The enzyme has only alanine as N-terminal amino acid and its N-terminal sequence shows a significant degree of homology with tryptophan 2-monooxygenase from Pseudomonas syringae pv. savastanoi. The pH optimum for the stability of the native enzyme is 5, similar to that of the barley leaf enzyme. Calorimetric analysis shows a single two-state transition at pH 6 with Tm 49.8 degrees. At pH 5 the thermal stability is increased by more than 14 degrees. Amine oxidation products, delta 1-pyrroline and diazabicyclononane, are competitive inhibitors of PAO activity (apparent Ki = 400 and 100 microM respectively). Moreover these compounds improve the thermal stability of the enzyme. N1-Acetylspermine, which is a good substrate for mammalian PAO, acts as a non-competitive inhibitor for the plant enzyme.     

Mammalian DNA ligases. Catalytic domain and size of DNA ligase I.

DNA ligase I is the major DNA ligase activity in proliferating mammalian cells. The protein has been purified to apparent homogeneity from calf thymus. It has a monomeric structure and a blocked N-terminal residue. DNA ligase I is a 125-kDa polypeptide as estimated by sodium dodecyl sulfate-gel electrophoresis and by gel chromatography under denaturing conditions, whereas hydrodynamic measurements indicate that the enzyme is an asymmetric 98-kDa protein. Immunoblotting with rabbit polyclonal antibodies to the enzyme revealed a single polypeptide of 125 kDa in freshly prepared crude cell extracts of calf thymus. Limited digestion of the purified DNA ligase I with several reagent proteolytic enzymes generated a relatively protease-resistant 85-kDa fragment. This domain retained full catalytic activity. Similar results were obtained with partially purified human DNA ligase I. The active large fragment represents the C-terminal part of the intact protein, and contains an epitope conserved between mammalian DNA ligase I and yeast and vaccinia virus DNA ligases. The function of the N-terminal region of DNA ligase I is unknown.     

Properties of herpes simplex virus DNA polymerase and characterization of its associated exonuclease activity.

Herpes simplex virus (HSV) DNA polymerase was isolated on a large-scale from African green monkey kidney cells infected with HSV type 1 (HSV-1) strain Angelotti. After DNA-cellulose chromatography the enzyme showed a specific activity of 48,000 units/mg protein. Three major single polypeptides with molecular weights of 144,000, 74,000 and 29,000 were copurified with the enzyme activity at the DNA-cellulose ste. By its chromatographic behavior and by template studies, the HSV DNA polymerase activity was clearly distinguishable from cellular alpha, beta and gamma DNA polymerase activities. Two exonucleolytic activities were found in the DNA-cellulose enzyme preparation. The main exonucleolytic activity, which degraded both single-stranded and double-stranded DNA to deoxynucleoside 5'-monophosphates, was separated by subsequent velocity sedimentation. The remaining exonucleolytic activity was not separable from the HSV DNA polymerase by several chromatographic steps and by velocity sedimentation at high ionic strength. This novel exonuclease and HSV DNA polymerase were equally sensitive both to phosphonoacetic acid and Zn2+ ions, inhibitors of the viral polymerase. Similar to the 3'-to-5'-exonuclease of procaryotic DNA polymerases and mammalian DNA polymerase delta, the HSV-polymerase-associated exonuclease catalyzed the removal of 3'-terminal nucleotides from the primer/template as well as the template-dependent conversion of deoxynucleoside triphosphates to monophosphates.     

DNA synthesis and dRPase activities of polymerase beta are both essential for single-nucleotide patch base excision repair in mammalian cell extracts

In mammalian cells the majority of altered bases in DNA are processed through a single-nucleotide patch base excision repair mechanism. Base excision repair is initiated by a DNA glycosylase that removes a damaged base and generates an abasic site (AP site). This AP site is further processed by an AP endonuclease activity that incises the phosphodiester bond adjacent to the AP site and generates a strand break containing 3'-OH and 5'-sugar phosphate ends. In mammalian cells, the 5'-sugar phosphate is removed by the AP lyase activity of DNA polymerase beta (Pol beta). The same enzyme also fills the gap, and the DNA ends are finally rejoined by DNA ligase. We measured repair of oligonucleotide substrates containing a single AP site in cell extracts prepared from normal and Pol beta-null mouse cells and show that the reduced repair in Pol beta-null extracts can be complemented by addition of purified Pol beta. Using this complementation assay, we demonstrate that mutated Pol beta without dRPase activity is able to stimulate long patch BER. Mutant Pol beta deficient in DNA synthesis, but with normal dRPase activity, does not stimulate repair in Pol beta-null cells. However, under conditions where we measure base excision repair accomplished exclusively through a single-nucleotide patch BER, neither dRPase nor DNA synthesis mutants of Pol beta alone, or the two together, were able to complement the repair defect. These data suggest that the dRPase and DNA synthesis activities of Pol beta are coupled and that both of these Pol beta functions are essential during short patch BER and cannot be efficiently substituted by other cellular enzymes.     

Expression of active rat DNA polymerase beta in Escherichia coli

A recombinant plasmid for expression of rat DNA polymerase beta was constructed in a plasmid/phage chimeric vector, pUC118, by an oligonucleotide-directed mutagenesis technique. The insert contained a 1005 bp coding sequence for the whole rat DNA polymerase beta. The recombinant plasmid was designed to use the regulatory sequence of Escherichia coli lac operon and the initiation ATG codon for beta-galactosidase as those for DNA polymerase beta. The recombinant clone, JMp beta 5, obtained by transfection of E. coli JM109 with the plasmid, produced high levels of DNA polymerase activity and a 40-kDa polypeptide that were not detected in JM109 cell extract. Inducing this recombinant E. coli with isopropyl beta-thiogalactopyranoside (IPTG) yielded amounts of 40-kDa polypeptide as high as 19.3% of total protein. Another recombinant clone, JMp beta 2-1, which was constructed by an oligonucleotide-directed mutagenesis to use the second ATG codon for the initiation codon, thus deleting the first 17 amino acid residues from the amino terminus, produced neither high DNA polymerase activity nor the 40-kDa polypeptide. The evidence suggests that this amino-terminal structure is important for stability of this enzyme in E. coli. The DNA polymerase was purified to homogeneity from the IPTG-induced JMp beta 5 cells by fewer steps than the procedure for purification of DNA polymerase beta from animal cells. The properties of this enzyme in activity, chromatographic behavior, size, antigenicity, and also lack of associated nuclease activity were indistinguishable from those of DNA polymerase beta purified from rat cells, indicating the identity of the overproduced DNA polymerase in the JMp beta 5 and the rat DNA polymerase beta.