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)

Mutations in the Drosophila mus308 gene confer specific hypersensitivity to DNA-cross-linking agents as a consequence of defects in DNA repair. The mus308 gene is shown here to encode a 229-kDa protein in which the amino-terminal domain contains the seven conserved motifs characteristic of DNA and RNA helicases and the carboxy-terminal domain shares over 55% sequence similarity with the polymerase domains of prokaryotic DNA polymerase I-like enzymes. This is the first reported member of this family of DNA polymerases in a eukaryotic organism, as well as the first example of a single polypeptide with homology to both DNA polymerase and helicase motifs. Identification of a closely related gene in the genome of Caenorhabditis elegans suggests that this novel polypeptide may play an evolutionarily conserved role in the repair of DNA damage in eukaryotic organisms.
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PMID:Molecular cloning of Drosophila mus308, a gene involved in DNA cross-link repair with homology to prokaryotic DNA polymerase I genes. 881 90

Herpesviruses are a very interesting model for studying DNA replication in eukaryotic systems since they encode most of the proteins required for this process. These include a protein that specifically binds to the virus origin of DNA synthesis, a single-stranded DNA binding protein, an heterotrimeric helicase-primase and an heterodimeric DNA polymerase holoenzyme. Although the virus genome contains three origins of DNA synthesis, replication proceeds through the generation of high molecular mass concatemeric replicative intermediates reminiscent of rolling circles. In addition, herpesviruses are highly recombinogenic and are useful models to study homologous recombination. Homologous recombination and replication of the virus genome appear to be tightly coupled and interrelated processes.
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PMID:Herpes simplex virus type 1 replication and recombination. 882 76

The RecG protein of Escherichia coli is a DNA helicase that promotes branch migration of the Holliday junctions. We found that overproduction of RecG protein drastically decreased copy numbers of ColE1-type plasmids, which require R-loop formation between the template DNA and a primer RNA transcript (RNA II) for the initiation of replication. RecG efficiently inhibited in vitro ColE1 DNA synthesis in a reconstituted system containing RNA polymerase, RNase HI and DNA polymerase I. RecG promoted dissociation of RNA II from the R-loop in a manner that required ATP hydrolysis. These results suggest that overproduced RecG inhibits the initiation of replication by prematurely resolving the R-loops formed at the replication origin region of these plasmids with its unique helicase activity. The possibility that RecG regulates the initiation of a unique mode of DNA replication, oriC-independent constitutive stable DNA replication, by its activity in resolving R-loops is discussed.
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PMID:ATP-dependent resolution of R-loops at the ColE1 replication origin by Escherichia coli RecG protein, a Holliday junction-specific helicase. 900 81

We report the nucleotide sequence of a 31-kb segment at the left genome end of bovine herpesvirus-1 (BHV-1) and show that it comprises 19 different open reading frames (ORFs), including seven which have been described previously (circ, dUTPase, UL49.5, alpha TIF, VP8, glycoprotein C, and ribonucleotide reductase small subunit). The new sequence resulted in a correction at the C-terminus of glycoprotein C. All 19 ORFs exhibited strong amino acid sequence homology to the gene products of other alphaherpesviruses. The BHV-1 ORFs were arranged colinearly with the prototype sequence of herpes simplex virus 1 (HSV-1) in the range of the UL54 to UL37 genes. No BHV-1 homologs of the HSV-1 UL56, UL55, and UL45 genes were identified. The BHV-1 circ gene was the only gene without a HSV-1 counterpart. The additional ORFs 1 and 2 found at the left genome end of equine herpesvirus-1 (EHV-1) were absent in BHV-1. Among the newly sequenced BHV-1 ORFs are homologs of ICP27 (UL54), glycoprotein K (UL53), helicase-primase (UL52), DNA polymerase accessory protein (UL42), ribonucleotide reductase large subunit (UL39), and several virion proteins (UL49, UL46, UL43, UL41, UL38, UL37), most of which are strongly conserved in all herpesviruses. The possible functions of the proteins encoded within the sequenced region are assessed and features found are discussed.
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PMID:Gene contents in a 31-kb segment at the left genome end of bovine herpesvirus-1. 901 Sep 99

Untransformed cells have been proposed to require a protein homologous to SV40 large tumor antigen (TAg) which functions as a component of the replicase complex during the initiation of DNA synthesis. By definition, this should be a phosphoprotein which interacts with the retinoblastoma protein (pRb) in G0 or early G1, and is capable of binding to and potentiating the activity of DNA polymerase alpha (pol alpha). This protein should also be an ATP-dependent helicase which interacts with the single-stranded DNA (ssDNA) binding protein, RP-A. Because of these requirements, a TAg homologous protein could be expected to contain epitopes with amino acid sequences similar to those of TAg at critical functional sites, such as ATP, pRb and pol alpha binding sites. TAg and a putative cellular homolog of TAg, DNA pol alpha accessory protein (alpha AP), were compared for pRb and pol alpha interaction, and for immunological identity. The analyses utilized immunoaffinity-purified TAg and pRb from a baculovirus expression system, and DNA pol alpha/primase and alpha AP chromatographically isolated from a mouse lymphocytic leukemia cell line. Monoclonal antibodies specific for the pol alpha or pRb binding sites on TAg interacted with alpha AP strongly enough to be employed for immunoaffinity purification of alpha AP. Anti-pRb and anti-TAg reciprocally coimmunoprecipitated pRb bound to TAg and pRb bound to alpha AP. The functional consequences of pol alpha interaction with TAg or alpha AP in the presence or absence of pRb was determined using pol alpha nucleotide incorporation assays. alpha AP exhibited the capacity to stimulate pol alpha activity, a capacity which was diminished in the presence of pRb. Lastly, TAg and alpha AP independently co-purified with pol alpha through a multi-step chromatographic protocol. These data indicate that a pol alpha accessory protein, alpha AP, exhibits functional and immunological similarities to SV40 TAg, suggest that alpha AP is involved in regulation of the initiation of DNA synthesis, and support the proposal that alpha AP may be a normal cell protein homologous to SV40 large T antigen.
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PMID:A DNA polymerase alpha accessory protein exhibits structural and functional similarities to SV40 large tumor antigen. 906 21

Bacteriophage T7 gene 2.5 single-stranded DNA-binding protein and gene 4 DNA helicase together promote pairing of two homologous DNA molecules and subsequent polar branch migration (Kong, D., and Richardson, C. C. (1996) EMBO J. 15, 2010-2019). In this report, we show that gene 2.5 protein is not required for the initiation or propagation of strand transfer once a joint molecule has been formed between the two DNA partners, a reaction that is mediated by the gene 2.5 protein alone. A mutant gene 2.5 protein, gene 2.5-Delta21C protein, lacking 21 amino acid residues at its C terminus, cannot physically interact with gene 4 protein. Although it does bind to single-stranded DNA and promote the formation of joint molecule via homologous base pairing, subsequent strand transfer by gene 4 helicase is inhibited by the presence of the gene 2.5-Delta21C protein. Bacteriophage T4 gene 32 protein likewise inhibits T7 gene 4 protein-mediated strand transfer, whereas Escherichia coli single-stranded DNA-binding protein does not. The 63-kDa gene 4 protein of phage T7 is also a DNA primase in that it catalyzes the synthesis of oligonucleotides at specific sequences during translocation on single-stranded DNA. We find that neither the rate nor extent of strand transfer is significantly affected by concurrent primer synthesis. The bacteriophage T4 gene 41 helicase has been shown to catalyze polar branch migration after the T4 gene 59 helicase assembly protein loads the helicase onto joint molecules formed by the T4 UvsX and gene 32 proteins (Salinas, F., and Kodadek, T. (1995) Cell 82, 111-119). We find that gene 32 protein alone forms joint molecules between partially single-stranded homologous DNA partners and that subsequent branch migration requires this single-stranded DNA-binding protein in addition to the gene 41 helicase and the gene 59 helicase assembly protein. Similar to the strand transfer reaction, strand displacement DNA synthesis catalyzed by T4 DNA polymerase also requires the presence of gene 32 protein in addition to the gene 41 and 59 proteins.
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PMID:Role of the bacteriophage T7 and T4 single-stranded DNA-binding proteins in the formation of joint molecules and DNA helicase-catalyzed polar branch migration. 907 62

The herpes simplex virus type 1 (HSV) single-stranded DNA-binding protein (SSB, ICP8) stimulates the viral DNA polymerase (Pol) on an oligonucleotide-primed single-stranded DNA template. This stimulation is non-specific since other SSBs also increase Pol activity. However, only ICP8 was stimulatory when Pol activity was dependent upon priming by the viral helicase-primase complex. ICP8 also specifically stimulated the primer synthesis and ATPase activities of the helicase-primase. The mechanism of stimulation was different from that of Pol; helicase-primase stimulation required much lower amounts of ICP8 than the amount that saturates the DNA and optimally stimulates Pol. Furthermore, ICP8 did not act by removing secondary structure as stimulation also occurred on homopolymer templates. While the UL8 component of the helicase-primase is not required for enzymatic activities by a subassembly of the UL5 and UL52 proteins, only the holoenzyme (UL5/8/52) was stimulated by ICP8. These results identify a unique, functional interaction between the ICP8 SSB and the helicase-primase complex, mediated by the UL8 subunit.
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PMID:A functional interaction of ICP8, the herpes simplex virus single-stranded DNA-binding protein, and the helicase-primase complex that is dependent on the presence of the UL8 subunit. 912 59

Although the overall picture of HCMV DNA synthesis appears typical of the herpesviruses, some novel features are emerging. Six herpesvirus-group-common genes encode proteins that likely constitute the replication fork machinery, including a two-subunit DNA polymerase, a helicas-primase complex and a single-stranded DNA-binding protein. No homolog of the herpes simplex virus origin-binding helicase is evident, but at least one additional HCMV protein of unknown function, pUL84, appears to be required for initiation. Replication initiates within or near the large and structurally complex lytic-phase replicator, ori-Lyt, near the center of UL. Recent findings suggest that ori-Lyt-mediated initiation of DNA synthesis occurs through a mechanism distinct from that employed by herpes simplex virus.
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PMID:The human cytomegalovirus genes and proteins required for DNA synthesis. 913 47

The gene 4 proteins of bacteriophage T7 provide both primase and helicase activities at the replication fork. Efficient DNA replication requires that the functions of the gene 4 protein be coordinated with the movement of the T7 DNA polymerase. We show that a carboxyl-terminal domain of the gene 4 protein is required for interaction with T7 DNA polymerase during leading strand DNA synthesis. The carboxyl terminus of the gene 4 protein is highly acidic: of the 17 carboxyl-terminal amino acids 7 are negatively charged. Deletion of the coding region for these 17 residues results in a gene 4 protein that cannot support the growth of T7 phage. The purified mutant gene 4 protein has wild-type levels of both helicase and primase activities; however, DNA synthesis catalyzed by T7 DNA polymerase on a duplex DNA substrate is stimulated by this mutant protein to only about 5% of the level of synthesis obtained with wild-type protein. The mutant gene 4 protein can form hexamers and bind single-stranded DNA, but as determined by native PAGE analysis, the protein cannot form a stable complex with the DNA polymerase. The mutant gene 4 protein can prime DNA synthesis normally, indicating that for lagging strand synthesis a different set of helicase/primase-DNA polymerase interactions are involved. These findings have implications for the mechanisms coupling leading and lagging strand DNA synthesis at the T7 replication fork.
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PMID:The acidic carboxyl terminus of the bacteriophage T7 gene 4 helicase/primase interacts with T7 DNA polymerase. 921 86

The herpes simplex virus type 1 (HSV-1) UL8 DNA replication protein is a component of a trimeric helicase-primase complex. Sixteen UL8-specific monoclonal antibodies (MAbs) were isolated and characterized. In initial immunoprecipitation experiments, one of these, MAb 804, was shown to coprecipitate POL, the catalytic subunit of the HSV-1 DNA polymerase, from extracts of insect cells infected with recombinant baculoviruses expressing the POL and UL8 proteins. Coprecipitation of POL was dependent on the presence of UL8 protein. Rapid enzyme-linked immunosorbent assays (ELISAs), in which one protein was bound to microtiter wells and binding of the other protein was detected with a UL8- or POL-specific MAb, were developed to investigate further the interaction between the two proteins. When tested in the ELISAs, five of the UL8-specific MAbs consistently inhibited the interaction, raising the possibility that these antibodies act by binding to epitopes at or near a site(s) on UL8 involved in its interaction with POL. The epitopes recognized by four of the inhibitory MAbs were approximately located by using a series of truncated UL8 proteins expressed in mammalian cells. Three of these MAbs recognized an epitope near the C terminus of UL8, which was subjected to fine mapping with a series of overlapping peptides. The C-terminal peptides were then tested in the ELISA for their ability to inhibit the POL-UL8 interaction: the most potent exhibited a 50% inhibitory concentration of approximately 5 microM. Our findings suggest that the UL8 protein may be involved in recruiting HSV-1 DNA polymerase into the viral DNA replication complex and also identify a potential new target for antiviral therapy.
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PMID:The catalytic subunit of the DNA polymerase of herpes simplex virus type 1 interacts specifically with the C terminus of the UL8 component of the viral helicase-primase complex. 926 56


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