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

---

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

We report the purification and characterization of a novel DNA helicase from calf thymus tissue. This enzyme partially copurifies with DNA polymerase epsilon* through many of the chromatographic procedures used to isolate it. The enzyme contains an intrinsic DNA-dependent ATPase activity. It can displace short oligonucleotides annealed to long single stranded substrates, in an ATP-dependent reaction. Use of this assay indicates that the DNA helicase translocates in a 3' to 5' direction with respect to the substrate strand to which it is bound. Maximal efficiency of displacement is accomplished by hydrolysis of (d)ATP as cofactor, however, (d)CTP can also be utilized resulting in a 5-fold decrease in the level of displacement. Displacement activity is enhanced by the presence of saturating amounts of Escherichia coli single stranded DNA-binding protein, not affected by the presence of phage T4 gene 32 protein, and inhibited by human replication factor A. The DNA helicase has a molecular mass of approximately 104 kDa as measured by denaturing gel electrophoresis, and an S value of 5.4 obtained from glycerol gradient sedimentation. Direct [alpha-32P]ATP cross-linking labels a protein of molecular mass approximately 105 kDa, providing further evidence that this polypeptide contains the helicase active site. In view of the differences in the properties of this helicase from four others recently identified in calf and designated A through D, we propose the name helicase E.
...
PMID:A novel DNA helicase from calf thymus. 132 24

In spite of the fact that a DNA helicase is clearly required for the predominantly leading-strand synthesis occurring during mammalian mtDNA replication, no such activity has heretofore been identified. We report the characterization of a mammalian mitochondrial DNA helicase isolated from bovine brain tissue. The sucrose gradient-purified mitochondria in which the activity was detected had less than 1 part in 2500 nuclear contamination according to Western blot analysis using nuclear- and mitochondrial-specific probes. Mitochondrial protein fractionation by DEAE-Sephacel chromatography yielded a DNA helicase activity dependent upon hydrolysis of ATP or dATP but not other NTPs or dNTPs. The mitochondrial helicase unwound 15- and 20-base oligonucleotides but was unable to unwind 32-base or longer oligonucleotides, and the polarity of the unwinding is 3'-to-5' with respect to the single-stranded portion of the partial duplex DNA substrate. This direction of unwinding would place the bovine mitochondrial helicase on the template strand ahead of DNA polymerase gamma during mtDNA replication, a situation analogous to that of the Rep helicase of Escherichia coli during leading-strand DNA synthesis of certain bacteriophages.
...
PMID:DNA helicase from mammalian mitochondria. 132 59

A functional interaction between DNA helicase E and DNA polymerase epsilon from calf thymus has been detected which results in the extension of an upstream 3' OH through a downstream primer to the end of a synthetic template. DNA synthesis resulting in full-length extension products was dependent on the addition of DNA helicase E and hydrolysis of ATP, suggesting that displacement of the downstream primer was required. Identical reactions using DNA polymerases alpha and delta in place of DNA polymerase epsilon showed no full-length products dependent on helicase E, indicating that polymerases alpha and delta were incapable of functionally interacting with the helicase. The reaction leading to full-length extension products was time dependent and dependent on the concentration of added polymerase epsilon and helicase E. Exonucleolytic degradation of the downstream primer, or ligation of the downstream primer to the upstream 3' OH, were not responsible for the full-length products observed. Displacement of the downstream primer by DNA helicase E was not affected by the addition of polymerase epsilon to the reactions. Template dilution experiments demonstrated that DNA polymerase epsilon and helicase E were acting in concert to perform displacement synthesis. Additional evidence for functional coordination was obtained by demonstration that DNA helicase E stimulated DNA polymerase epsilon in a standard DNA synthetic assay using dA3000.dT16 as the template-primer. The results presented are consistent with the hypothesis that DNA helicase E and DNA polymerase epsilon are capable of coordinated activities that result in displacement synthesis. A functional interaction of this sort may be involved at the eukaryotic replication fork or in DNA repair.
...
PMID:DNA helicase E and DNA polymerase epsilon functionally interact for displacement synthesis. 132 8

We have previously proposed that DNA polymerase alpha-primase provides short RNA-DNA precursors below 40 nucleotides (DNA primers), several of which assemble into an Okazaki piece after intervening RNA has been removed and the gaps have been filled by DNA polymerase delta (or epsilon) (T. Nethanel, S. Reisfeld, G. Dinter-Gottlieb, and G. Kaufmann, J. Virol. 62:2867-2873, 1988; T. Nethanel and G. Kaufmann, J. Virol. 64:5912-5918, 1990). In this report, we confirm and extend these conclusions by studying the effects of deoxynucleoside triphosphate (dNTP) concentrations and the presence of ATP on the occurrence, dynamics, and configuration of DNA primers in simian virus 40 replicative intermediate DNA. We first show that these parameters are not significantly affected by a 10-fold increase in dNTP precursor concentrations. We then demonstrate that Okazaki piece synthesis can be arrested at the level of DNA primers by ATP depletion. The arrested DNA primers faced short gaps of 10 to 20 nucleotides at their 3' ends and were progressively chased into Okazaki pieces when ATP was restored. ATP could not be substituted in this process by adenosine-5'-O-(3-thiotriphosphate) or adenyl-imidodiphosphate. The chase was interrupted by aphidicolin but not by butylphenyl-dGTP. The results implicate an ATP-requiring factor in the switch between the two DNA polymerases engaged in Okazaki piece synthesis. They also suggest that the replication fork advances by small, DNA primer-size increments.
...
PMID:Assembly of simian virus 40 Okazaki pieces from DNA primers is reversibly arrested by ATP depletion. 132 83

A helicase-like DNA unwinding activity was found in highly purified fractions of the calf thymus single-stranded DNA binding protein (ctSSB), also known as replication protein A (RP-A) or replication factor A (RF-A). This activity depended on the hydrolysis of ATP or dATP, and used CTP with a lower efficiency. ctSSB promoted the homologous DNA polymerase alpha to perform DNA synthesis on double-stranded templates containing replication fork-like structures. The rate and amount of DNA synthesis was found to be dependent on the concentration of ctSSB. At a 10-fold mass excess of ctSSB over double-stranded DNA, products of 200-600 nucleotides in length were obtained. This comprises or even exceeds the length of a eukaryotic Okazaki fragment. The ctSSB-associated DNA helicase activity is most likely a distinct protein rather than an inherent property of SSB, as inferred from titration experiments between SSB and DNA. The association of a helicase with SSB and the stimulatory action of this complex to the DNA polymerase alpha-catalyzed synthesis of double-stranded DNA suggests a cooperative function of the three enzymatic activities in the process of eukaryotic DNA replication.
...
PMID:A complex between replication factor A (SSB) and DNA helicase stimulates DNA synthesis of DNA polymerase alpha on double-stranded DNA. 133 Jun 89

The dnaB gene of Escherichia coli encodes an essential DNA replication enzyme. Fueled by the energy derived from the hydrolysis of ATP to ADP+P(i), this enzyme unwinds double-stranded DNA in advance of the DNA polymerase. While doing so, it intermittently stimulates primase to synthesize an RNA primer for an Okazaki fragment. To better understand the structural basis of these and other aspects of DnaB function, we have initiated a study of mutant DnaB proteins. Here, we report the purification and characterization of a mutant DnaB protein (RC231) containing cysteine in place of arginine at residue 231. The mutant protein attains a stable, properly folded structure that allows association of six promoters to form a hexamer, as is also true for wild-type DnaB. Further, the mutant protein interacts with ATP, the nonhydrolyzable ATP analog adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S), ADP, and poly(dT), and it stimulates primase action. It is, however, profoundly deficient in ATP hydrolysis, helicase activity, and replication activity at the chromosomal origin of replication. In addition, while general priming reactions with wild-type DnaB and ATP elicited the synthesis of short primers, reactions with DnaB and ATP gamma S or with RC231 and either ATP or ATP gamma S stimulated the synthesis of significantly longer primers. On the basis of these observations, we suggest that primase interacts directly with DnaB throughout primer synthesis during general priming, until dissociation of DnaB from DNA or ATP hydrolysis by DnaB disrupts the interaction and leads to primer termination.
...
PMID:Purification and characterization of a mutant DnaB protein specifically defective in ATP hydrolysis. 133 41

The elongation of primed DNA templates by DNA polymerase delta and DNA polymerase epsilon requires the action of two accessory proteins, proliferating cell nuclear antigen and activator 1 (A1, also called replication factor C). A1 is an enzyme that contains five different subunits (145, 40, 38, 37, and 36.5 kDa). In this paper, we describe the isolation of the gene encoding the 37-kDa subunit from HeLa cells. This gene was cloned, sequenced, and overexpressed in Escherichia coli. The amino acid sequence shows a high degree of homology to the 40-kDa subunit of A1; they both contain the identical ATP-binding motif, but in contrast to the bacterial expressed 40-kDa protein, the 37-kDa expressed protein did not bind ATP. Both the 37- and 40-kDa proteins share substantial homology with the phage T4 gene 44 protein and to a lesser extent with the tau and gamma subunits of the E. coli DNA polymerase III holoenzyme. Polyclonal antibodies against the bacterially expressed 37- and 40-kDa proteins do not crossreact and are specific in their interaction. Antibodies against the 37-kDa protein maximally inhibited (by 50%) the A1-dependent synthesis of DNA by DNA polymerase delta; antibodies against the 40-kDa protein quantitatively inhibited the same reaction. When A1-dependent synthesis of DNA was partially inhibited by antibodies against the 40-kDa subunit, the addition of antibodies against the 37-kDa subunit inhibited DNA synthesis to a greater extent than the anti-37-kDa antibody alone. These results suggest that both the 37- and 40-kDa subunits of A1 are required for the biological role of A1 and that they may function differently in this process.
...
PMID:Studies of the cloned 37-kDa subunit of activator 1 (replication factor C) of HeLa cells. 135 77

By using a complementation assay that enabled DNA polymerase delta and DNA polymerase epsilon to replicate a singly-DNA primed M13 DNA in the presence of proliferating cell nuclear antigen (PCNA) and Escherichia coli single-stranded DNA binding protein (SSB), we have purified from calf thymus in a five step procedure a multipolypeptide complex with molecular masses of polypeptides of 155, 70, 60, 58, 39 (doublet), 38 (doublet) and 36 kDa. The protein is very likely replication factor C (Tsurimoto, T. and Stillman, B. (1989) Mol. Cell. Biol. 9, 609-619). This conclusion is based on biochemical and physicochemical data and the finding that it contains a DNA stimulated ATPase which is under certain conditions stimulated by PCNA. Together RF-C, PCNA and ATP convert DNA polymerases delta and epsilon to holoenzyme forms, which were able to replicate efficiently SSB-covered singly-DNA primed M13 DNA. Calf thymus RF-C could form a primer recognition complex on a 3'-OH primer terminus in the presence of calf thymus PCNA and ATP. Holoenzyme complexes of DNA polymerase delta and epsilon could be isolated suggesting that these enzymes directly interact with the auxiliary proteins in a similar way. Under optimal replication conditions on singly-DNA primed M13 DNA the DNA synthesis rate of DNA polymerase delta was higher than of DNA polymerase epsilon. Based on these functional date possible roles of these two DNA polymerases in eukaryotic DNA replication are discussed.
...
PMID:Calf thymus RF-C as an essential component for DNA polymerase delta and epsilon holoenzymes function. 135 54

DNA polymerase-beta was purified from Novikoff hepatoma and used as an antigen in an in vitro immunization system to produce monoclonal antibodies. These reagents surprisingly showed cross-reactivity to a number of proteins, including several DNA polymerases. Nearly all of these proteins possess nucleotide binding sites, which suggested the potential value of using the monoclonals to elucidate structure-function relationships within polymerase-beta. Furthermore, these antibodies were able to partially neutralize (40-50%) polymerase-beta activity, and this effect could be blocked by dNTP1 but not by dNMP or rNTP. The limited neutralization phenomenon is at least partially explained by the weak binding affinity of these antibodies. Scatchard analysis of immunoprecipitation data predicted a Kd of 1.8 x 10(-8) M. Epitope mapping studies showed that the region of polymerase-beta recognized by one of the monoclonal antibodies is within residues 235-335, and sequence homology studies indicated that the epitope is probably located in the region of amino acids 283-320. At least a portion of this area, namely residues 301-308 and 311-315, appears to be part of a nucleotide binding domain which has sequence homology with a portion of the highly conserved ATP binding site in adenylate kinase.
...
PMID:Structure-function analysis of DNA polymerase-beta using monoclonal antibodies: identification of a putative nucleotide binding domain. 138 Aug 29

In order to clarify the biological activities of (-)-oxetanocin G, and (-)-oxetanocin A and its carbocyclic analogue, (-)-carboxetanocin G, the inhibitory effects of triphosphate derivatives of these compounds (OXT-GTP, OXT-ATP, and C-OXT-GTP) on eukaryotic and viral DNA polymerases were examined. DNA polymerase alpha purified from calf thymus was weakly inhibited by OXT-GTP and OXT-ATP but strongly by C-OXT-GTP, the Ki value being 0.22 microM. On the other hand, rat DNA polymerase beta was not affected by these analogues. DNA polymerase gamma purified from bovine testes was very weakly inhibited by OXT-GTP and OXT-ATP, but not by C-OXT-GTP. DNA polymerase from herpes simplex virus type-II (HSV-II) was strongly inhibited by all three analogues, the Ki values ranging from 0.5 to 1.0 microM. Human immunodeficiency virus-encoded reverse transcriptase (HIV RT) was also strongly inhibited by these three analogues, the Ki value of C-OXT-GTP being slightly smaller than that of OXT-GTP or OXT-ATP. Analysis of products synthesized on singly primed M13 single-stranded DNA by DNA polymerase alpha, HSV-II DNA polymerase or HIV RT in the presence of the analogues revealed that OXT-GTP and C-OXT-GTP were incorporated into DNA and caused chain termination mainly at sites one or two nucleotides beyond the cytosine bases on the template.
...
PMID:Inhibitory effects of triphosphate derivatives of oxetanocin G and related compounds on eukaryotic and viral DNA polymerases and human immunodeficiency virus reverse transcriptase. 138 92


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---