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)

The DNA replication system of bacteriophage T4 serves as a relatively simple model for the types of reactions and protein-protein interactions needed to carry out and coordinate the synthesis of the leading and lagging strands of a DNA replication fork. At least 10 phage-encoded proteins are required for this synthesis: T4 DNA polymerase, the genes 44/62 and 45 polymerase accessory proteins, gene 32 single-stranded DNA binding protein, the genes 61, 41, and 59 primase-helicase, RNase H, and DNA ligase. Assembly of the polymerase and the accessory proteins on the primed template is a stepwise process that requires ATP hydrolysis and is strongly stimulated by 32 protein. The 41 protein helicase is essential to unwind the duplex ahead of polymerase on the leading strand, and to interact with the 61 protein to synthesize the RNA primers that initiate each discontinuous fragment on the lagging strand. An interaction between the 44/62 and 45 polymerase accessory proteins and the primase-helicase is required for primer synthesis on 32 protein-covered DNA. Thus it is possible that the signal for the initiation of a new fragment by the primase-helicase is the release of the polymerase accessory proteins from the completed adjacent fragment.
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PMID:Protein-protein interactions at a DNA replication fork: bacteriophage T4 as a model. 131 Sep 46

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.
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PMID:A novel DNA helicase from calf thymus. 132 24

The herpes simplex virus (HSV) type 1 helicase-primase is a three-protein complex, consisting of a 1:1:1 association of UL5, UL8, and UL52 gene products (J.J. Crute, T. Tsurumi, L. Zhu, S. K. Weller, P. D. Olivo, M. D. Challberg, E. S. Mocarski, and I. R. Lehman, Proc. Natl. Acad. Sci. USA 86:2186-2189, 1989). We have purified this complex, as well as a subcomplex consisting of UL5 and UL52 proteins, from insect cells infected with baculovirus recombinants expressing the appropriate gene products. In confirmation of previous reports, we find that whereas UL5 alone has greatly reduced DNA-dependent ATPase activity, the UL5/UL52 subcomplex retains the activities characteristic of the heterotrimer: DNA-dependent ATPase activity, DNA helicase activity, and the ability to prime DNA synthesis on a poly(dT) template. We also found that the primers made by the subcomplex are equal in length to those synthesized by the UL5/UL8/UL52 complex. In an effort to uncover a role for UL8 in HSV DNA replication, we have developed a model system for lagging-strand synthesis in which the primase activity of the helicase-primase complex is coupled to the activity of the HSV DNA polymerase on ICP8-coated single-stranded M13 DNA. Using this assay, we found that the UL8 subunit of the helicase-primase is critical for the efficient utilization of primers; in the absence of UL8, we detected essentially no elongation of primers despite the fact that the rate of primer synthesis on the same template is undiminished. Reconstitution of lagging-strand synthesis in the presence of UL5/UL52 was achieved by the addition of partially purified UL8. Essentially identical results were obtained when Escherichia coli DNA polymerase I was substituted for the HSV polymerase/UL42 complex. On the basis of these findings, we propose that UL8 acts to increase the efficiency of primer utilization by stabilizing the association between nascent oligoribonucleotide primers and template DNA.
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PMID:The UL8 subunit of the herpes simplex virus helicase-primase complex is required for efficient primer utilization. 132 Dec 75

Epstein-Barr virus (EBV) utilizes a completely different mode of DNA replication during the lytic cycle than that employed during latency. The latency origin of replication, ori-P, which functions in the replication of the latent episomal form of the EBV genome, requires only a single virally encoded protein, EBNA-1, for its activity. During the lytic cycle, a separate origin, ori-Lyt, is utilized. Relatively little is known about the trans-acting proteins involved in ori-Lyt replication. We established a cotransfection-replication assay to identify EBV genes whose products are required for replication of ori-Lyt. In this assay, a BamHI-H plasmid containing ori-Lyt was replicated in Vero cells cotransfected with the BamHI-H target, the three EBV lytic-cycle transactivators Zta, Rta, and Mta, and the EBV genome provided in the form of a set of six overlapping cosmid clones. By removing individual cosmids from the cotransfection mixture, we found that only three of the six cosmids were necessary for ori-Lyt replication. Subcloning of the essential cosmids led to the identification of six EBV genes that encode replication proteins. These genes and their functions (either known or predicted on the basis of sequence comparison with herpes simplex virus) are BALF5, the DNA polymerase; BALF2, the single-stranded DNA-binding protein homolog; BMRF1, the DNA polymerase processivity factor; BSLF1 and BBLF4, the primase and helicase homologs; and BBLF2/3, a potential homolog of the third component of the helicase-primase complex. In addition, ori-Lyt replication in this cotransfection assay was also dependent on one or more genes provided by the EBV SalI-F fragment and on the three lytic-cycle transactivators Zta, Rta, and Mta.
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PMID:trans-acting requirements for replication of Epstein-Barr virus ori-Lyt. 132 Dec 85

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.
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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.
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PMID:DNA helicase E and DNA polymerase epsilon functionally interact for displacement synthesis. 132 8

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.
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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.
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PMID:Purification and characterization of a mutant DnaB protein specifically defective in ATP hydrolysis. 133 41

Bacteriophage T4 DNA replication initiates from origins at early times of infection and from recombinational intermediates as the infection progresses. Plasmids containing cloned T4 origins replicate during T4 infection, providing a model system for studying origin-dependent replication. In addition, recombination-dependent replication can be analyzed by using cloned nonorigin fragments of T4 DNA, which direct plasmid replication that requires phage-encoded recombination proteins. We have tested in vivo requirements for both plasmid replication model systems by infecting plasmid-containing cells with mutant phage. Replication of origin and nonorigin plasmids strictly required components of the T4 DNA polymerase holoenzyme complex. Recombination-dependent plasmid replication also strictly required the T4 single-stranded DNA-binding protein (gene product 32 [gp32]), and replication of origin-containing plasmids was greatly reduced by 32 amber mutations. gp32 is therefore important in both modes of replication. An amber mutation in gene 41, which encodes the replicative helicase of T4, reduced but did not eliminate both recombination- and origin-dependent plasmid replication. Therefore, gp41 may normally be utilized for replication of both plasmids but is apparently not required for either. An amber mutation in gene 61, which encodes the T4 RNA primase, did not eliminate either recombination- or origin-dependent plasmid replication. However, plasmid replication was severely delayed by the 61 amber mutation, suggesting that the protein may normally play an important, though nonessential, role in replication. We deleted gene 61 from the T4 genome to test whether the observed replication was due to residual gp61 in the amber mutant infection. The replication phenotype of the deletion mutant was identical to that of the amber mutant. Therefore, gp61 is not required for in vivo T4 replication. Furthermore, the deletion mutant is viable, demonstrating that the gp61 primase is not an essential T4 protein.
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PMID:Plasmid models for bacteriophage T4 DNA replication: requirements for fork proteins. 143 1

(+)-CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis. Previous studies have shown that the potent cytotoxic and antitumor activities of (+)-CC-1065 are due to the ability of this compound to covalently modify DNA. (+)-CC-1065 reacts with duplex DNA to form a (N3-adenine)-DNA adduct which lies in the minor groove of DNA overlapping with a five base-pair region. As a consequence of covalent modification with (+)-CC-1065, the helix bends into the minor groove and also undergoes winding and stiffening. In the studies described here, we have constructed templates for helicase-catalyzed unwinding of DNA that contain site-directed (+)-CC-1065 and analogue DNA adducts. Using these templates we have shown that (+)-CC-1065 and select synthetic analogues, which have different levels of cytotoxicity, all produce a significant inhibition of unwinding of a 3'-tailed oligomer duplex by helicase II when the displaced strand is covalently modified. However, the extent of helicase II inhibition is much more significant for (+)-CC-1065 and an analogue which also produced DNA winding when the winding effects are transmitted in the opposite direction to the helicase unwinding activity. This observed pattern of inhibition of helicase-catalyzed unwinding of drug-modified templates was the same for a 3'-T-tail, for different duplex region sequences, and with the Escherichia coli rep protein. Unexpectedly, the gel mobility of the displaced drug-modified single strand was dependent on the species of drug attached to the DNA. Last, strand displacement by helicase II coupled to primer extension by E. coli DNA polymerase I showed the same pattern of inhibition when the lagging strand was covalently modified. In addition, the presence of helicase II on single-stranded regions of templates caused the premature termination of primer extension by DNA polymerase. These results are discussed from the perspective that (+)-CC-1065 and its analogues have different effects on DNA structure, and these resulting structural changes in DNA molecules are related to the different in vivo biological consequences caused by these drug molecules.
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PMID:Structure-activity relationships of (+)-CC-1065 analogues in the inhibition of helicase-catalyzed unwinding of duplex DNA. 158 57


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