Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have recently shown that DNA containing homopolymeric A-T base pairs can form a parallel-stranded intramolecular duplex [van de Sande et al. (1988) Science (Washington, D.C.) 241, 551-557]. In the present paper, we demonstrate that parallel-stranded DNA can also be formed in unconstrained bimolecular DNA of appropriate sequence homology. Three deoxyoligonucleotides, a 21-mer [dCCCATATATATTTTTTTTCCC], a ps-15-mer [dTATATATAAAAAAAA], and an aps-15-mer [dAAAAAAAATATATAT], have been synthesized. Annealing of 21-mer and aps-15-mer results in the formation of a conventional antiparallel duplex (aps); however, the combination of 21-mer and ps-15-mer forms a duplex in which the two strands are in a parallel orientation (ps). The parallel-stranded structure was established from the following criteria: (i) The parallel-stranded structure shows a 1:1 stoichiometry of the constituent strands. (ii) Gel electrophoretic mobility of the ps and aps duplexes are similar under native conditions. (iii) Spectroscopic properties of the ps duplex are characteristics for a base-paired structure but are different from the aps structure. (iv) Both duplexes undergo a thermally induced helix to coil transition; however, the melting temperature for the ps duplex is 22 degrees C lower. (v) The minor groove binding drug Hoechst 33258 shows a reduced affinity for the ps compared to the aps duplex. (vi) The parallel-stranded duplex is not a substrate for DNA Escherichia coli polymerase I (Klenow fragment) or AMV reverse transcriptase. Parallel-stranded DNA can exist under normal solution conditions, but competition experiments show it to be thermodynamically less favorable than the conventional antiparallel form.
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PMID:Relative stability of parallel- and antiparallel-stranded duplex DNA. 246 57

A DNA-binding protein has been identified that recognizes runs of deoxyadenines and/or deoxythymines (dA/dT sequences) and purified from a chromatographic fraction containing the multiprotein DNA polymerase alpha-primase complex of HeLa cells by successive steps of chromatography on oligo(dT)-cellulose and Q-Sepharose. Polyacrylamide gel electrophoresis of the purified dA/dT sequence-binding protein in the presence of NaDodSO4 showed a single protein band of 62 kDa. Nitrocellulose filter binding assays using homopolydeoxynucleotides indicated that the purified protein preferentially binds to dA/dT sequences in single-stranded or duplex DNAs. Gel mobility shift assays with a variety of DNAs showed that the purified protein specifically binds to a fragment of simian virus 40 DNA containing the minimal (core) origin for replication. The binding occurred in a protein-dependent manner and in the presence of a vast excess of competing DNAs lacking the simian virus replication origin. The origin binding was reduced, however, when DNA fragments from simian virus 40 deletion mutants containing deletions within the 17-base-pair A + T-rich tract in the core DNA replication origin were used in the assays. These results indicate that the dA/dT sequence-binding protein preferentially binds to the 17-base-pair A + T-rich tract and suggest a possible role for the protein in the initiation of DNA replication.
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PMID:Sequence recognition protein for the 17-base-pair A + T-rich tract in the replication origin of simian virus 40 DNA. 253 62

Distamycin A, a polypeptide antibiotic, binds to dA.dT-rich regions in the minor groove of B-DNA. By virtue of its nonintercalating binding, distamycin acts as a potent inhibitor of the synthesis of DNA both in vivo and in vitro. Here we report that distamycin paradoxically stimulates Escherichia coli DNA polymerase I (pol I), its large (Klenow) fragment, and bacteriophage T4 DNA polymerase to copy oligo(dA).poly(dT) in vitro. It is found that distamycin increases the maximum velocity (Vmax) of the extension of the oligo(dA) primer by pol I without affecting the Michaelis constant (Km) of the primer. Gel electrophoresis of the extended primer indicates that the antibiotic specifically increases the rate of addition of the first three dAMP residues. Lastly, in the presence of both distamycin and the oligo(dT)-binding protein factor D, which increases the processivity of pol I, a synergistic stimulation of polymerization is attained. Taken together, these results suggest that distamycin stimulates synthesis by increasing the rate of initiation of oligo(dA) extension. The stimulatory effect of distamycin is inversely related to the stability of the primer-template complex. Thus, maximum stimulation is exerted at elevated temperatures and with shorter oligo(dA) primers. That distamycin increases the thermal stability of [32P](dA)9.poly(dT) is directly demonstrated by electrophoretic separation of the hybrid from dissociated [32P](dA)9 primer. It is proposed that by binding to the short primer-template duplex, distamycin stabilizes the oligo(dA).poly(dT) complex and, therefore, increases the rate of productive initiations of synthesis at the primer terminus.
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PMID:Distamycin paradoxically stimulates the copying of oligo(dA).poly(dT) by DNA polymerases. 281 66

Previous in vivo studies have indicated that (dT-dC)n.(dG-dA)n tracts (referred to here as (TC)n.(GA)n), which are widely dispersed in vertebrate genomes, may serve as pause or arrest signals for DNA replication and amplification. To determine whether these repeat elements act as stop signals for DNA replication in vitro, single stranded DNAs including (TC)n or (GA)n tracts of various lengths, were prepared by cloning such tracts into phage M13 vectors, and were replicated with the Klenow fragment of the E. coli DNA polymerase I, or with the calf thymus DNA polymerase alpha, by extension of an M13 primer. Gel electrophoresis of the reaction products revealed that the replication was specifically arrested around the middle of both (TC)n and (GA)n tracts of n greater than or equal to 16. However, whereas in the (TC)n tracts the arrests were less prominent at pH = 8.0 than at pH = 6.5-7.5, and were completely eliminated at pH = 8.5, the arrests in the (GA)n tracts were stronger at the higher pH values. These results, and previous data, suggest that the arrests were caused by formation of unusual DNA structures, possibly triple helices between partially replicated (TC)n or (GA)n tracts, and unreplicated portions of these sequences.
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PMID:(dT-dC)n and (dG-dA)n tracts arrest single stranded DNA replication in vitro. 292 74

A single-stranded DNA-binding protein (SSB) affinity column was prepared by optimizing the coupling of Escherichia coli single-stranded DNA-binding protein to Affi-Gel 10. The bound SSB retained its ability to specifically bind single-stranded DNA. When nuclease-treated cell extracts were incubated with the SSB beads overnight at 4 degrees C, a major protein of Mr = 25,000 was bound. At shorter incubation times, two additional proteins of Mr = 32,000 and 36,000 were also detected. In the absence of nuclease treatment, eight additional proteins ranging from Mr = 14,000 to 160,000 also bound to the affinity column. The major Mr = 25,000 protein has been shown to be a folded chromosome-associated protein. Its binding to SSB is strongly enhanced by the addition of DNA polymerase III or DNA polymerase III holoenzyme.
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PMID:Interaction of a folded chromosome-associated protein with single-stranded DNA-binding protein of Escherichia coli, identified by affinity chromatography. 304 82

The beta subunit of Escherichia coli DNA polymerase III holoenzyme binds Mg2+. Reacting beta with fluoresceinmaleimide (FM) resulted in one label per beta monomer with full retention of activity. Titration of FM-beta with Mg2+ resulted in a saturable 11% fluorescence enhancement. Analysis indicated that there was one noncooperative magnesium binding site per beta monomer with a dissociation constant of 1.7 mM. Saturable fluorescence enhancement was also observed when titration was with Ca2+ or spermidine(3+) but not with the monovalent cations Na+ and K+. The Mg2+-induced fluorescence enhancement was specific for FM-beta and was not observed with FM-glutathione, dimethoxystilbenemaleimide-beta, or pyrenylmaleimide-beta. Gel filtration studies indicated that the beta dimer-monomer dissociation occurred at physiologically significant beta concentrations and that the presence of 10 mM Mg2+ shifted the dimer-monomer equilibrium to favor monomers. Both the gel-filtered dimers and the gel-filtered monomers were active in the replication assay. These and other results suggested that the fluorescence increase which accompanies beta dissociation is due to a relief from homoquenching of FM when the beta dimer dissociates into monomers.
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PMID:The dimer of the beta subunit of Escherichia coli DNA polymerase III holoenzyme is dissociated into monomers upon binding magnesium(II). 304 97

The role of exonuclease activity in trans-lesion DNA replication with Escherichia coli DNA polymerase III holoenzyme was investigated. RecA protein inhibited the 3'----5' exonuclease activity of the polymerase 2-fold when assayed in the absence of replication and had no effect on turnover of dNTPs into dNMPs. In contrast, single-stranded DNA-binding protein, which had no effect on the exonuclease activity in the absence of replication, showed a pronounced 7-fold suppression of the 3'----5' exonuclease activity during replication. The excision of incorporated dNMP alpha S residues from DNA by the 3'----5' exonuclease activity of DNA polymerase III holoenzyme was inhibited 10-20-fold; still no increase in bypass of pyrimidine photodimers was observed. Thus, in agreement with our previous results in which the exonuclease activity was inhibited at the protein level (Livneh, Z. (1986) J. Biol. Chem. 261, 9526-9533), inhibition at the DNA level also did not increase bypass of photodimers. Fractionation of the replication mixture after termination of DNA synthesis on a Bio-Gel A-5m column under conditions which favor polymerase-DNA binding yielded a termination complex which could perform turnover of dNTPs into dNMPs. Adding challenge-primed single-stranded DNA to the complex yielded a burst of DNA synthesis which was promoted most likely by DNA polymerase III holoenzyme molecules transferred from the termination complex to the challenge DNA thus demonstrating the instability of the polymerase-DNA association. Addition of a fresh sample of DNA polymerase III holoenzyme to purified termination products, which consist primarily of partially replicated molecules with nascent chains terminated at UV lesions, did not result in any net DNA synthesis as expected. However, reactivation of lesion-terminated primers was achieved by pretreatment with a 3'----5' exonuclease which excised 200 nucleotides or more, generating new 3'-OH termini located away from the UV lesions. When these exonuclease-treated products were subjected to a second round of replication, an increased level of DNA synthesis was observed including additional bypass of photodimers. These results suggest the possibility that 3'----5' exonuclease processing might be required at least transiently during one of the stages of trans-lesion DNA replication, which is believed to be the mechanism of SOS-targeted mutagenesis.
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PMID:The role of exonucleolytic processing and polymerase-DNA association in bypass of lesions during replication in vitro. Significance for SOS-targeted mutagenesis. 305 41

Conditions for efficient replication in vitro of mitochondrial DNA L strand into H strand products have been established. Gel electrophoresis and hybridization analyses of the products show that neosynthesized H strands are progressively elongated from the D-loop region, and some of them are synthesized as full-length molecules. Evidence for initiation of these H strands de novo is presented. In contrast, there is no detectable L strand synthesis in vitro in this system. This may prove useful for analyzing the distinct molecular mechanisms operating at OH and OL. Use of specific inhibitors indicates that DNA synthesis in the mitochondrial lysate in vitro requires DNA polymerase gamma. These observations support the conclusion that replication in vitro in this system closely resembles the first steps of mitochondrial DNA replication in vivo.
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PMID:DNA synthesis in a mitochondrial lysate of Xenopus laevis oocytes. H strand replication in vitro. 311 61

For the specific purification of eukaryotic DNA-dependent DNA polymerase alpha, we prepared two novel affinity resins bearing 5-(E)-(4-aminostyryl) araUTP as a ligand. One of them was araUTP-Sepharose 4B which was coupled directly with the ligand and the other was araUTP-Affi-Gel 10 which was coupled with the ligand through a spacer. No DNA polymerase alpha-primase activity from cherry salmon (Oncorhynchus masou) testes was bound on the araUTP-Sepharose 4B in all cases examined. On the other hand, the araUTP-Affi-Gel 10 retains this enzyme activity when poly(dA) or poly(dA)-oligo(dT)12-18 is present. The retained enzyme activity was sharply eluted around 100-mM KCl concentrations as a single peak, and this fraction showed a specific activity of about 170,000 units/mg as alpha-polymerase activity. The highly purified DNA polymerase alpha-primase isolated using the araUTP-Affi-Gel 10 contained only three polypeptides, which showed Mr values of 120,000, 62,000, and 58,000, respectively, as judged using sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
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PMID:AraUTP-Affi-Gel 10: a novel affinity absorbent for the specific purification of DNA polymerase alpha-primase. 321 43

Yeast cells from a wild type or protease-deficient strain were lysed in the absence or presence of protease inhibitors and the extracts analyzed by analytical high pressure liquid chromatography on diethylaminoethyl silica gel. Conditions that inhibited protease action caused elution of a novel DNA polymerase activity at a position in the gradient distinct from the elution positions of both DNA polymerase I and II. In large scale purifications, this DNA polymerase, called DNA polymerase III, copurified with a single-stranded DNA dependent 3'-5' exonuclease activity, exonuclease III, to near homogeneity. Glycerol gradient centrifugation partially dissociated the complex to yield two peaks of exonuclease III activity, one at 7.7 S together with the DNA polymerase, and one at 4.0 S without polymerase activity. Gel filtration indicated that the complex has a molecular mass greater than 400 kDa. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the complex consists of several subunits: 140, 62, 55, and 53 kilodaltons, some of which may be proteolysis products. The exonuclease component of the complex can excise single nucleotide mismatches providing a base-paired primer-template which can be elongated by the DNA polymerase. Under replication conditions, the complex exhibits a measurable turnover rate of dTTP to dTMP and it contains no primase activity. The enzymatic activities of the 3'-5' exonuclease are consistent with a proofreading function during in vivo DNA replication. A second exonuclease activity, exonuclease IV, separated from the complex late in the purification scheme. It degrades both single-stranded and double-stranded DNA in the 5'----3' direction.
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PMID:DNA polymerase III from Saccharomyces cerevisiae. I. Purification and characterization. 327 61


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