Isolation of DNA polymerase beta from human placenta and study of its substrate specificity

A method of purification of DNA polymerase beta with a specific activity of 1300 units/mg from human placenta was developed. The enzyme preparations do not contain any other DNA polymerase activities and any nuclease contaminations degrading nucleic acids. On the basis of analysis of several standard parameters we conclude that the purified enzyme is polymerase beta. The optimal conditions of polymerization were established, and a comparison of the relative rates of polymerization with various template-primer complexes was carried out. Activated DNA was shown to be the optimal substrate in the presence of MgCl2, and poly(dA).oligo(dT) in the presence of MnCl2. The activation energies of polymerization for different template-primers were estimated.     

Mammalian base excision repair and DNA polymerase beta

OBJECTIVE: To describe six dogs with congenital abnormalities involving the portal vein, caudal vena cava, or both. ANIMALS: Six client-owned dogs with congenital interruption of the portal vein or the caudal vena cava, or both. METHODS: Portal vein and caudal vena cava anatomy was evaluated by contrast radiography and visualization at surgery. Vascular casts or plastinated specimens were obtained in three animals. RESULTS: Portal blood shunted into the caudal vena cava in four dogs and the left hepatic vein in one. Two of these five dogs also had interruption of the caudal vena cava with continuation as azygous vein, as did an additional dog, in which the portal vein was normally formed. Portal vein interruption was present in 5 of 74 (6.8%) dogs with congenital portosystemic shunts evaluated at the Veterinary Teaching Hospital during the study period. CONCLUSIONS: Serious malformations of the abdominal veins were present in more than 1 in 20 dogs with single congenital portosystemic shunts. CLINICAL RELEVANCE: Veterinarians involved in diagnosis and surgery for portosystemic shunts should be aware of these potential malformations, and portal vein continuity should be evaluated in all dogs before attempting shunt attenuation.     

Purification and characterization of DNA ligase III from bovine testes. Homology with DNA ligase II and vaccinia DNA ligase

Mammalian cell nuclei contain three biochemically distinct DNA ligases. In the present study we have found high levels of DNA ligase I and DNA ligase III activity in bovine testes and have purified DNA ligase III to near homogeneity. The high level of DNA ligase III suggests a role for this enzyme in meiotic recombination. In assays measuring the fidelity of DNA joining, we detected no significant differences between DNA ligases II and III, whereas DNA ligase I was clearly a more faithful enzyme and was particularly sensitive to 3' mismatches. Amino acid sequences of peptides derived from DNA ligase III demonstrated that this enzyme, like DNA ligase II, is highly homologous with vaccinia DNA ligase. The absence of unambiguous differences between homologous peptides from DNA ligases II and III (10 pairs of peptides, 136 identical amino acids) indicates that these enzymes are either derived from a common precursor polypeptide or are encoded from the same gene by alternative splicing. Based on similarities in amino acid sequence and biochemical properties, we suggest that DNA ligases II and III, Drosophila DNA ligase II, and the DNA ligases encoded by the pox viruses constitute a distinct family of DNA ligases that perform specific roles in DNA repair and genetic recombination.     

Increased activity and fidelity of DNA polymerase beta on single-nucleotide gapped DNA

DNA polymerase beta (pol beta) is an error-prone polymerase that plays a central role in mammalian base excision repair. To better characterize the mechanisms governing rat pol beta activity, we examined polymerization on synthetic primer-templates of different structure. Steady-state kinetic analyses revealed that the catalytic efficiency of pol beta (kcat/Km,dNTPapp) is strongly influenced by gap size and the presence of a phosphate group at the 5'-margin of the gap. pol beta exhibited the highest catalytic efficiency on 5'-phosphorylated 1-nucleotide gapped DNA. This efficiency was >/=500 times higher than on non-phosphorylated 1-nucleotide and 6-nucleotide (with or without PO4) gapped DNAs and 2,500 times higher than on primer-template with no gaps. The nucleotide insertion fidelity of pol beta, as judged by its ability to form G-N mispairs, was also higher (10-100 times) on 5'-phosphorylated single-nucleotide gapped DNA compared with the other DNA substrates studied. These data suggest that a primary function of mammalian pol beta is to fill 5'-phosphorylated 1-nucleotide gaps.     

The fidelity of DNA polymerase beta during distributive and processive DNA synthesis

During base excision repair, DNA polymerase beta fills 1-6-nucleotide gaps processively, reflecting a contribution of both its 8- and 31-kDa domains to DNA binding. Here we report the fidelity of pol beta during synthesis to fill gaps of 1, 5, 6, or >300 nucleotides. Error rates during distributive synthesis by recombinant rat and human polymerase (pol) beta with a 390-base gap are similar to each other and to previous values with pol beta purified from tissues. The base substitution fidelity of human pol beta when processively filling a 5-nucleotide gap is similar to that with a 361-nucleotide gap, but "closely-spaced" substitutions are produced at a rate at least 60-fold higher than for distributive synthesis. Base substitution fidelity when filling a 1-nucleotide gap is higher than when filling a 5-nucleotide gap, suggesting a contribution of the 8-kDa domain to the dNTP binding pocket and/or a difference in base stacking or DNA structure imposed by pol beta. Nonetheless, 1-nucleotide gap filling is inaccurate, even generating complex substitution-addition errors. Finally, the single-base deletion error rate during processive synthesis to fill a 6-nucleotide gap is indistinguishable from that of distributive synthesis to fill a 390-nucleotide gap. Thus the mechanism of processivity by pol beta does not allow the enzyme to suppress template misalignments.     

Excision of deoxyribose phosphate residues by DNA polymerase beta during DNA repair

Eukaryotic DNA polymerase beta (pol beta) can catalyze DNA synthesis during base excision DNA repair. It is shown here that pol beta also catalyzes release of 5'-terminal deoxyribose phosphate (dRP) residues from incised apurinic-apyrimidinic sites, which are common intermediate products in base excision repair. The catalytic domain for this activity resides within an amino-terminal 8-kilodalton fragment of pol beta, which comprises a distinct structural domain of the enzyme. Magnesium is required for the release of dRP from double-stranded DNA but not from a single-stranded oligonucleotide. Analysis of the released products indicates that the excision reaction occurs by beta-elimination rather than hydrolysis.     

Poly(ADP-ribose) polymerase stimulates DNA polymerase alpha by physical association

The direct effect of the eukaryotic nuclear DNA-binding protein poly(ADP-ribose) polymerase on the activity of DNA polymerase alpha was investigated. Homogenously purified poly(ADP-ribose) polymerase (5 to 10 micrograms/ml) stimulated the activity of immunoaffinity-purified calf or human DNA polymerase alpha by about 6 to 60-fold in a dose-dependent manner. It had no effect on the activities of DNA polymerase beta, DNA polymerase gamma, and primase, indicating that its effect is specific for DNA polymerase alpha. Apparently, poly(ADP-ribosyl)ation of DNA polymerase alpha was not necessary for the stimulation. The stimulatory activity is due to poly(ADP-ribose) polymerase itself since it was immunoprecipitated with a monoclonal antibody directed against poly(ADP-ribose) polymerase. Kinetic analysis showed that, in the presence of poly(ADP-ribose) polymerase, the saturation curve for DNA template primer became sigmoidal; at very low concentrations of DNA, it rather inhibited the reaction in competition with template DNA, while, at higher DNA doses, it greatly stimulated the reaction by increasing the Vmax of the reaction. By the automodification of poly(ADP-ribose) polymerase, however, both the inhibition at low DNA concentration and the stimulation at high DNA doses were largely lost. Furthermore, stimulation by poly(ADP-ribose) polymerase could not be attributed to its DNA-binding function alone since its fragment, containing only the DNA-binding domain, could not exert full stimulatory effect on DNA polymerase, as of the intact enzyme. Poly(ADP-ribose) polymerase is co-immunoprecipitated with DNA polymerase alpha, using anti-DNA polymerase alpha antibody, clearly showing that poly(ADP-ribose) polymerase may be physically associated with DNA polymerase alpha. In a crude extract of calf thymus, a part of poly(ADP-ribose) polymerase activity existed in a 400-kDa, as well as, a larger 700-kDa complex containing DNA polymerase alpha, suggesting the existence in vivo of a complex of these two enzymes.     

Optical mapping of DNA polymerase I action and products

Single molecule approaches to the characterization of biochemical systems offer an intrinsically simple and direct approach to address difficult, previously unyielding problems. Optically based approaches have recently been used to construct high resolution, ordered restriction maps from a variety of clone types. Advancements in surface technologies have enabled the reliable elongation and fixation of large DNA molecules onto specially derivatized substrates with retention of biochemical accessibility. In this study, the addition of fluorescently labeled nucleotides to surface-mounted DNA molecules by the action of DNA polymerase I is investigated using fluorescence microscopy to image individual template molecules. Molecules undergoing nick translation and containing only a few fluorochromes are readily imaged. These novel results suggest that surface-bound molecules may serve as a substrate for a broad range of enzymatic actions, and may offer new routes to analysis when coupled to advanced imaging techniques.     

The inhibitory action of fatty acids on DNA polymerase beta

Ten cases of subependymal giant cell astrocytoma associated with sclerosis tuberosa were reevaluated in order to assess their phenotyping and biologic features. All tumours were multifocal, located within lateral ventricles, often overlying the head of the caudate nucleus or protruding into the third ventricle. The phenotype of SGCA disclosed a complex pattern: giant cells were GFAP positive, some of them were stained with antibodies against neurofilament and NSE. Ultrastructurally, the cells of SGCA contained frequent dense bodies, numerous intermediate filaments and microtubules. Biologically SGCA is not malignant, although its appearance may suggest otherwise. No patient had an apparent recurrence within 3-5 years of observation.     

Human DNA polymerase beta recognizes single-stranded DNA using two different binding modes

Interactions between the human DNA polymerase beta (pol beta) and a single-stranded (ss) DNA have been studied using the quantitative fluorescence titration technique. Examination of the fluorescence increase of the poly(dA) etheno-derivative (poly(depsilonA)) as a function of the binding density of pol beta-nucleic acid complexes reveals the existence of two binding phases. In the first high affinity phase, pol beta forms a complex with a ssDNA in which 16 nucleotides are occluded by the enzyme. In the second phase, transition to a complex where the polymerase occludes only 5 nucleotides occurs. Thus, human pol beta binds a ssDNA in two binding modes, which differ in the number of occluded nucleotide residues. We designate the first complex as (pol beta)16 and the second as (pol beta)5 binding modes. The analyses of the enzyme binding to ssDNA have been performed using statistical thermodynamic models, which account for the existence of the two binding modes of the enzyme, cooperative interactions, and the overlap of potential binding sites. The importance of the discovery that human pol beta binds a ssDNA, using different binding modes, for the possible mechanistic model of the functioning of human pol beta, is discussed.     

A genetic system to identify DNA polymerase beta mutator mutants

Hemagglutinins were determined in six species of mosquitoes that are susceptible and refractory to Brugia malayi (Filarioidea: Nematoda). High titers of hemagglutinins were found in the salivary gland extract and in the body fluid of a completely refractory species, Aedes taeniorhynchus, and in partially refractory species, Anopheles quadrimculatus but low levels of hemagglutinins were also present in the body fluid of Aedes aegypti (Black-eye, Liverpool strain), a susceptible species. Hemagglutinating activity was not found in the other three completely refractory species of mosquitoes, Culex quinquefasciatus, Culex nigripalpus, and Aedes albopictus in which blood coagulated rapidly after ingestion. High titers of hemagglutinins in the salivary glands of Ae. taeniorhynchus and An. quadrimaculatus facilitated rapid movement of sheathed microfilariae from the midgut to the hemocoel. It is suggested that high titers of hemagglutinins present in the hemocoel bound to the glycoconjugates with exposed carbohydrate moieties present on the microfilarial sheaths and developing abnormal larvae (L1) in the thoracic muscle cells. These hemagglutinin-bound glycoconjugates formed capsules that subsequently stimulated the immune response and resulted in melanization of microfilarial sheaths and sheathed microfilariae in the hemocoel and intracellularly developing abnormal L1 in the thoracic muscles. Only minimal encapsulation and melanization of B. malayi microfilariae was observed in the hemocoel of the other four species of mosquitoes that lacked hemagglutinins in the salivary glands. The results suggest that tissue specific hemagglutinins are one of several factors of vector susceptibility/refractoriness through immune reactions (encapsulation, activation of prophenoloxidases).     

Purification and characterization of a new DNA polymerase from budding yeast Saccharomyces cerevisiae. A probable homolog of mammalian DNA polymerase beta

A new DNA polymerase activity was identified and purified to near homogeneity from extracts of mitotic and meiotic cells of the yeast Saccharomyces cerevisiae. This activity increased at least 5-fold during meiosis, and it was shown to be associated with a 68-kDa polypeptide as determined by SDS-polyacrylamide gel electrophoresis. This new DNA polymerase did not have any detectable 3'-->5' exonuclease activity and preferred small gapped DNA as a template-primer. The activity was inhibited by dideoxyribonucleoside 5'-triphosphates and N-ethylmaleimide but not by concentrations of aphidicolin which completely inhibit either DNA polymerases I (alpha), II (epsilon), or III (delta). Since no polypeptide(s) in the extensively purified DNA polymerase fractions cross-reacted with antibodies raised against yeast DNA polymerases I, II, and III, we called this enzyme DNA polymerase IV. The DNA polymerase IV activity increased at least 10-fold in a yeast strain overexpressing the gene product predicted from the YCR14C open-reading frame (identified on S. cerevisiae chromosome III and provisionally called POLX), while no activity was detected in a strain where POLX was deleted. These results strongly suggest that DNA polymerase IV is encoded by the POLX gene and is a probable homolog of mammalian DNA polymerase beta.     

2.3 A crystal structure of the catalytic domain of DNA polymerase beta

The crystal structure of the catalytic domain of rat DNA polymerase beta (pol beta) has been determined at 2.3 A resolution and refined to an R factor of 0.22. The mixed alpha/beta protein has three subdomains arranged in an overall U shape reminiscent of other polymerase structures. The folding topology of pol beta, however, is unique. Two divalent metals bind near three aspartic acid residues implicated in the catalytic activity. In the presence of Mn2+ and dTTP, interpretable electron density is seen for two metals and the triphosphate, but not the deoxythymidine moiety. The principal interaction of the triphosphate moiety is with the bound divalent metals.     

The thioredoxin binding domain of bacteriophage T7 DNA polymerase confers processivity on Escherichia coli DNA polymerase I

Sphingomonas yanoikuyae B1 is extremely versatile in its catabolic ability. An insertional mutant strain, S. yamoikuyae EK504, which is unable to grow on naphthalene due to the loss of 2-hydroxychromene-2-carboxylate isomerase activity, was utilized to investigate the role of this enzyme in the degradation of anthracene by S. yanoikuyae B1. Although EK504 is unable to grow on anthracene, this strain could transform anthracene to some extent. A metabolite in the degradation of anthracene by EK504 was isolated by high-pressure liquid chromatography (HPLC) and was identified as 6,7-benzocoumarin by UV-visible, gas-chromatographic, HPLC/mass-spectrometric, and 1H nuclear magnetic resonance spectral techniques. The identification of 6,7-benzocoumarin provides direct chemical and genetic evidence for the involvement of nahD in the degradation of anthracene by S. yanoikuyae B1.     

Recognition of sequence-directed DNA structure by the Klenow fragment of DNA polymerase I

Time-resolved fluorescence spectroscopy was used to investigate the influence of sequence-directed DNA structure upon the interaction between the Klenow fragment of DNA polymerase I and a series of defined oligonucleotide primer/templates. 17/27-mer (primer/template) oligonucleotides containing a dansyl fluorophore conjugated to a modified deoxyuridine residue within the primer strand were used as substrates for binding to Klenow fragment. The time-resolved fluorescence anisotropy decay of the dansyl probe was analyzed in terms of two local environments, either solvent-exposed or buried, corresponding to primer/templates positioned with the primer 3' terminus in the polymerase site or the 3'-5' exonuclease site of the enzyme, respectively. Equilibrium constants for partitioning of DNA between the two sites were evaluated from the anisotropy decay data for primer/templates having different (A + T)-rich sequences flanking the primer 3' terminus. Primer/templates with AAAATG/TTTTAC and CGATAT/GCTATA terminal sequences (the nucleotides on the left refer to the last six bases at the 3' end of the primer, and the nucleotides on the right are the corresponding bases in the template) were bound mostly at the polymerase site. The introduction of single mismatches opposite the primer 3' terminus of these DNA substrates increased their partitioning into the 3'-5' exonuclease site, in accord with the results of an earlier study [Carver, T.E., Hochstrasser, R.A., and Millar, D.P. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10670-10674]. In contrast, a primer/template with the terminal sequence CAATTT/GTTAAA, containing an A-tract element AATTT, exhibited a surprising preference for binding at the 3'-5' exonuclease site, despite the absence of mismatched bases in the DNA substrate. Interruption of the A-tract with a single AG step, to give the terminal sequence CAGTTT/GTCAAA, reversed the effect of the A-tract, causing the DNA to partition in favor of the polymerase site. Moreover, the presence of a single mismatch opposite the primer 3' terminus was also sufficient to reverse the effect of the A-tract, resulting in a distribution of DNA between polymerase and 3'-5' exonuclease sites that was similar to that observed for the other mismatched DNA substrates. Taken together, these results suggest that the A-tract adopts an unusual conformation that is disruptive to binding at the polymerase site. The effect of the A-tract on binding of DNA to the polymerase site is discussed in terms of the unusual helix structural parameters associated with these sequence elements and the difference between the local geometry of the A-tract and the conformation adopted by duplex DNA within the polymerase cleft. The results of this study show that in addition to base mismatches, Klenow fragment can also recognize irregularities in the helix geometry of perfectly base-paired DNA.     

The biochemical inhibition mode of bredinin-5''-monophosphate on DNA polymerase beta

PURPOSE: To determine electrocardiographic features associated with myocardial salvage following reperfusion therapy in patients with inferior myocardial infarction. PATIENTS AND METHODS: Ninety-two consecutive patients with acute inferior myocardial infarction were treated with reperfusion therapy in a tertiary care center. Several features were measured on the presenting electrocardiogram, including the presence or absence of ST depression in the chest leads and the total magnitudes of ST elevation or depression, and were then evaluated for their association with myocardial salvage. Myocardial salvage (% of left ventricle) was the difference between myocardium at risk and final infarct size. Tomographic myocardial perfusion imaging with technetium-99m sestamibi was performed acutely to measure myocardium at risk and repeated prior to hospital discharge to measure final infarct size. RESULTS: The amount of myocardium at risk of infarction in the 92 patients was 19.1%+/-11.3% (range 1% to 68%), and the final infarct size was 10.6%+/-10.0% (range 0% to 45%). Thus, myocardial salvage in the 92 patients was 8.5%+/-8.4% (range -11% to 35%) of the left ventricle, or 0.51+/-0.38 (range 0.0 to 1.0) when expressed as a fraction of the myocardium at risk (salvage index). The presence or absence of anterior ST depression was the only one of seven electrocardiographic variables that was associated with myocardial salvage. Myocardial salvage was significantly greater in patients with anterior ST depression compared with those without it (10.6%+/-9.0% versus 5.9%+/-6.7%, P=0.025). Myocardium at risk was significantly greater in patients with anterior ST depression compared with those without the depression (22.8%+/-12.2% versus 14.6%+/-8.3%, P=0.0006), and infarct size tended to be larger (12.1%+/-10.4% versus 8.7%+/-9.4%, P=0.10). Myocardial salvage as a fraction of the myocardium at risk (salvage index) was similar between the two patient groups (0.52+/-0.37 versus 0.50+/-0.39, P=NS). CONCLUSION: The presence of anterior ST depression during inferior myocardial infarction identifies a group of patients with the potential for greater myocardial salvage with reperfusion therapy. Such patients derive greater absolute benefit from reperfusion therapy because they have a larger amount of myocardium at risk, although their response to therapy (salvage index) is not intrinsically different.     

Rare association of human herpesvirus 6 DNA with human papillomavirus DNA in cervical smears of women with normal and abnormal cytologies

We investigated by nested PCR the possible association of human herpesvirus 6 (HHV-6) and human papillomavirus (HPV) genomes in the cervixes of 109 women with normal and abnormal cytological smears. HPV DNA was detected in 8.33% of 24 women with normal cytologies and in 41.1% of 85 women with abnormal cytologies; the proportion of HPV DNA was directly related to the severity of the lesions. HHV-6 DNA was found in only one patient, who had a cytological pattern of koilocytosis. The HHV-6 genome was classified by restriction enzyme analysis as variant B. The study indicates that detection of the HHV-6 genome in the cervixes of women with a wide spectrum of gynecological complaints is a rare event and rules out the possible association between HHV-6 and HPV genomes in cervical cancer lesions.     

Excision of C-4'-oxidized deoxyribose lesions from double-stranded DNA by human apurinic/apyrimidinic endonuclease (Ape1 protein) and DNA polymerase beta

Oxidative damage to DNA deoxyribose generates oxidized abasic sites (OAS) that may constitute one-third of ionizing radiation damage. The antitumor drug bleomycin produces exclusively OAS in the form of C-4-keto-C-1-aldehydes in unbroken DNA strands and 3'-phosphoglycolate esters terminating strand breaks. We investigated whether two human DNA repair enzymes can mediate OAS excision in vitro: Ape1 protein (the main human abasic endonuclease (also called Hap1, Apex, or Ref1)) and DNA polymerase beta, which carries out both the abasic excision and the resynthesis steps. We used a duplex oligonucleotide substrate with one main target for bleomycin-induced damage. Ape1 catalyzed effective incision at the C-4-keto-C-1-aldehyde sites at a rate that may be only a few-fold lower than incision of hydrolytic abasic sites at the same location. Consistent with several previous studies, Ape1 hydrolyzed 3'-phosphoglycolates 25-fold more slowly than C-4-keto-C-1-aldehydes. DNA polymerase beta excised the 5'-terminal OAS formed by Ape1 incision at a rate similar to its removal of unmodified abasic residues. Polymerase beta-mediated excision of 5'-terminal OAS was stimulated by Ape1 as it is for unmodified abasic sites. Escherichia coli Fpg (MutM) protein also excised 5'-terminal OAS, but in our hands, the RecJ protein did not. These observations help define mammalian pathways of OAS repair, point to interactions that might coordinate functional steps, and suggest that still unknown factors may contribute to removal of 3'-phosphoglycolate esters.