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)

Repair of DNA interstrand cross-links is a challenging problem for cells. Many human gene products influence sensitivity to DNA cross-linking agents, but the mechanisms of cross-link repair are unknown. In Drosophila melanogaster, the mus308 mutation leads to marked sensitivity to DNA cross-linking agents. The C-terminal portion of the Mus308 polypeptide encodes a DNA polymerase, whereas a putative DNA helicase is encoded by the N-terminal portion. As a step toward isolating proteins involved in DNA cross-link repair, we searched for mammalian genes similar to the DNA helicase portion of Mus308. Human and mouse homologs were isolated from cDNA expression libraries and designated HEL308. Human HEL308 is on chromosome 4q21 and encodes a polypeptide of 1101 amino acids. The protein was expressed in insect cells and purified. HEL308 is a single-stranded DNA-dependent ATPase and DNA helicase. Mutation of a highly conserved lysine to methionine in helicase domain I eliminated both activities. The protein readily displaces 20- to 40-mer duplex oligonucleotides. Displacement of longer substrates was less efficient but was stimulated by the single-stranded DNA-binding protein RPA. Activity was supported by ATP or dATP but not other nucleotide triphosphates. The enzyme translocates on DNA with 3' to 5' polarity and behaves as a multimer upon gel filtration.
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PMID:A human DNA helicase homologous to the DNA cross-link sensitivity protein Mus308. 1175 61

Bacteriophage phiKZ is a giant virus that efficiently infects Pseudomonas aeruginosa strains pathogenic to human and, therefore, it is attractive for phage therapy. We present here the complete phiKZ genome sequence and a preliminary analysis of its genome structure. The 280,334 bp genome is a linear, circularly permutated and terminally redundant, A+T-rich double-stranded DNA molecule. The phiKZ DNA has no detectable sequence homology to other viruses and microorganisms, and it does not contain NotI, PstI, SacI, SmaI, XhoI, and XmaIII endonuclease restriction sites. The genome has 306 open reading frames (ORFs) varying in size from 50 to 2237 amino acid residues. According to the orientation of transcription, ORFs are apparently organized into clusters and most have a clockwise direction. The phiKZ genome also encodes six tRNAs specific for Met (AUG), Asn (AAC), Asp (GAC), Leu (TTA), Thr (ACA), and Pro (CCA). A putative promoter sequence containing a TATATTAC block was identified. Most potential stem-loop transcription terminators contain the tetranucleotide UUCG loops. Some genes may be assigned as phage-encoded RNA polymerase subunits. Only 59 phiKZ gene products exhibit similarity to proteins of known function from a diversity of organisms. Most of these conserved gene products, such as dihydrofolate reductase, ribonucleoside diphosphate reductase, thymidylate synthase, thymidylate kinase, and deoxycytidine triphosphate deaminase are involved in nucleotide metabolism. However, no virus-encoded DNA polymerase, DNA replication-associated proteins, or single-stranded DNA-binding protein were found based on amino acid homology, and they may therefore be strongly divergent from known homologous proteins. Fifteen phiKZ gene products show homology to proteins of pathogenic organisms, including Mycobacterium tuberculosis, Haemophilus influenzae, Listeria sp., Rickettsia prowazakeri, and Vibrio cholerae that must be considered before using this phage as a therapeutic agent. The phiKZ coat contains at least 40 polypeptides, and several proteins are cleaved during virus assembly in a way similar to phage T4. Eleven phiKZ-encoded polypeptides are related to proteins of other bacteriphages that infect a variety of hosts. Among these are four gene products that contain a putative intron-encoded endonuclease harboring the H-N-H motif common to many double-stranded DNA phages. These observations provide evidence that phages infecting diverse hosts have had access to a common genetic pool. However, limited homology on the DNA and protein levels indicates that bacteriophage phiKZ represents an evolutionary distinctive branch of the Myoviridae family.
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PMID:The genome of bacteriophage phiKZ of Pseudomonas aeruginosa. 1191 76

We previously used a pattern recognition program for nucleic acids to detect sequences with the potential to form intrastrand triplexes. Potential intrastrand triplex (PIT) element families were found in Escherichia coli, Synechocystis sp. and Haemophilus influenza. We were particularly intrigued with the family found in E. coli, which contained 25 dispersed copies of a particular PIT sequence corresponding to the purine triplex motif. E. coli PIT elements appear to occur exclusively in non-coding regions. We now report biochemical experiments testing the interaction of E. coli PIT elements with polymerases and single-stranded DNA-binding protein (SSB). The elements were also tested in genetic experiments as promoters, transcription terminators, or replication pause sites in E. coli. We show that PIT elements display provocative characteristics in certain biochemical assays. When appropriately oriented, the elements block elongation by Taq DNA polymerase at 72 degrees C, but not elongation by T7 DNA polymerase at 37 degrees C. The G-rich strand of the E. coli PIT sequence folds into a form with reduced affinity for SSB. On the other hand, in vivo studies did not detect replication delays for conjugal transfer of episomes containing PIT elements. These sequences were shown not to act as promoters, but the presence of PIT elements in RNA leaders upstream of a coding region could strongly influence expression of the downstream gene. These effects were shown to be post-transcriptional and were solely dependent on the Watson-Crick stem-loop structure within the PIT element. Thus, although PIT element DNA displays unusual biochemical properties, it remains unknown how these elements arose, and why they persist in the E. coli genome.
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PMID:Functional studies of potential intrastrand triplex elements in the Escherichia coli genome. 1205 44

The mouse hypervariable minisatellite (MN) Pc-1 consists of tandem repeats of d(GGCAG) and flanked sequences. We have previously demonstrated that single-stranded d(GGCAG)(n) folds into the intramolecular folded-back quadruplex structure under physiological conditions. Because DNA polymerase progression in vitro is blocked at the repeat, the characteristic intramolecular quadruplex structure of the repeat, at least in part, could be responsible for the hypermutable feature of Pc-1 and other MNs with similar repetitive units. On the other hand, we have isolated six MN Pc-1 binding proteins (MNBPs) from nuclear extracts of NIH 3T3 cells. Here, we describe one of those MNBPs, MNBP-B, that binds to the single-stranded d(GGCAG)(n). Amino acid sequences of seven proteolytic peptide fragments of MNBP-B were determined, and the cDNA clones were isolated. MNBP-B was proven identical to the single-stranded DNA-binding protein, UP1. Recombinant UP1 bound to single-stranded d(GGCAG)(n) and other G-rich repetitive sequences, such as d(GTCAGG)(n) and d(GTTAGG)(n). In addition, UP1 was demonstrated by CD spectrum analysis to unfold the intramolecular quadruplex structure of d(GGCAG)(5) and d(TTAGGG)(4) and to abrogate the arrest of DNA synthesis at the d(GGG)(n) site. This ability of UP1 suggests that unfolding of quadruplex DNA is required for DNA synthesis processes.
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PMID:Unfolding of quadruplex structure in the G-rich strand of the minisatellite repeat by the binding protein UP1. 1223 55

Gene 2.5 of bacteriophage T7 is an essential gene that encodes a single-stranded DNA-binding protein. T7 phage with gene 2.5 deleted can grow only on Escherichia coli cells that express gene 2.5 from a plasmid. This complementation assay was used to screen for lethal mutations in gene 2.5. By screening a library of randomly mutated plasmids encoding gene 2.5, we identified 20 different single amino acid alterations in gene 2.5 protein that are lethal in vivo. The location of these essential residues within the three-dimensional structure of gene 2.5 protein assists in the identification of motifs in the protein. In this study we show that a subset of these alterations defines the dimer interface of gene 2.5 protein predicted by the crystal structure. Recombinantly expressed and purified gene 2.5 protein-P22L, gene 2.5 protein-F31S, and gene 2.5 protein-G36S do not form dimers at salt concentrations where the wild-type gene 2.5 protein exists as a dimer. The basis of the lethality of these mutations in vivo is not known because altered proteins retain the ability to bind single-stranded DNA, anneal complementary strands of DNA, and interact with T7 DNA polymerase.
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PMID:Essential amino acid residues in the single-stranded DNA-binding protein of bacteriophage T7. Identification of the dimer interface. 1237 53

The organization and proper assembly of proteins to the primer-template junction during DNA replication is essential for accurate and processive DNA synthesis. DNA replication in RB69 (a T4-like bacteriophage) is similar to those of eukaryotes and archaea and has been a prototype for studies on DNA replication and assembly of the functional replisome. To examine protein-protein interactions at the DNA replication fork, we have established solution conditions for the formation of a discrete and homogeneous complex of RB69 DNA polymerase (gp43), primer-template DNA, and RB69 single-stranded DNA-binding protein (gp32) using equilibrium fluorescence and light scattering. We have characterized the interaction between DNA polymerase and single-stranded DNA-binding protein and measured a 60-fold increase in the overall affinity of RB69 single-stranded DNA-binding protein (SSB) for template strand DNA in the presence of DNA polymerase that is the result of specific protein-protein interactions. Our data further suggest that the cooperative binding of the RB69 DNA polymerase and SSB to the primer-template junction is a simple but functionally important means of regulatory assembly of replication proteins at the site of action. We have also shown that a functional domain of RB69 single-stranded DNA-binding protein suggested previously to be the site of RB69 DNA polymerase-SSB interactions is dispensable. The data from these studies have been used to model the RB69 DNA polymerase-SSB interaction at the primer-template junction.
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PMID:Biochemical characterization of interactions between DNA polymerase and single-stranded DNA-binding protein in bacteriophage RB69. 1245 97

Gene 2.5 of bacteriophage T7 encodes a single-stranded DNA-binding protein that is essential for viral survival. Its crystal structure reveals a conserved oligosaccharide/oligonucleotide binding fold predicted to interact with single-stranded DNA. However, there is no experimental evidence to support this hypothesis. Recently, we reported a genetic screen for lethal mutations in gene 2.5 that we are using to identify functional domains of the gene 2.5 protein. This screen uncovered a number of mutations that led to amino acid substitutions in the proposed DNA binding domain. Three variant proteins, gp2.5-Y158C, gp2.5-K152E, and gp2.5-Y111C/Y158C, exhibit a decrease in binding affinity for oligonucleotides. A fourth, gp2.5-K109I, exhibits an altered mode of binding single-stranded DNA. A carboxyl-terminal truncation of gene 2.5 protein, gp2.5-Delta26C, binds single-stranded DNA 10-fold more tightly than the wild-type protein. The three altered proteins defective in single-stranded DNA binding cannot mediate the annealing of homologous DNA, whereas gp2.5-Delta26C mediates the reaction more effectively than does wild-type. Gp2.5-K109I retains this annealing ability, albeit slightly less efficiently. With the exception of gp2.5-Delta26C, all variant proteins form dimers in solution and physically interact with T7 DNA polymerase.
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PMID:The DNA binding domain of the gene 2.5 single-stranded DNA-binding protein of bacteriophage T7. 1249 73

Gene 5 protein (gp5) of bacteriophage T7 is a non-processive DNA polymerase, which acquires high processivity by binding to Escherichia coli thioredoxin. The gene 5 protein-thioredoxin complex (gp5/trx) polymerizes thousands of nucleotides before dissociating from a primer-template. We have engineered a disulfide linkage between the gene 5 protein and thioredoxin within the binding surface of the two proteins. The polymerase activity of the covalently linked complex (gp5-S-S-trx) is similar to that of gp5/trx on poly(dA)/oligo(dT). However, gp5-S-S-trx has only one third the polymerase activity of gp5/trx on single-stranded M13 DNA. gp5-S-S-trx has difficulty polymerizing nucleotides through sites of secondary structure on M13 DNA and stalls at these sites, resulting in lower processivity. However, gp5-S-S-trx has an identical processivity and rate of elongation when E. coli single-stranded DNA-binding protein (SSB protein) is used to remove secondary structure from M13 DNA. Upon completing synthesis on a DNA template lacking secondary structure, both complexes recycle intact, without dissociation of the processivity factor, to initiate synthesis on a new DNA template. However, a complex stalled at secondary structure becomes unstable, and both subunits dissociate from each other as the polymerase prematurely releases from M13 DNA.
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PMID:A covalent linkage between the gene 5 DNA polymerase of bacteriophage T7 and Escherichia coli thioredoxin, the processivity factor: fate of thioredoxin during DNA synthesis. 1269 31

The annealing of complementary strands of DNA is a vital step during the process of DNA replication, recombination, and repair. In bacteriophage T7-infected cells, the product of viral gene 2.5, a single-stranded DNA-binding protein, performs this function. We have identified a single amino acid residue in gene 2.5 protein, arginine 82, that is critical for its DNA annealing activity. Expression of gene 2.5 harboring this mutation does not complement the growth of a T7 bacteriophage lacking gene 2.5. Purified gene 2.5 protein-R82C binds single-stranded DNA with a greater affinity than the wild-type protein but does not mediate annealing of complementary strands of DNA. A carboxyl-terminal-deleted protein, gene 2.5 protein-Delta26C, binds even more tightly to single-stranded DNA than does gene 2.5 protein-R82C, but it anneals homologous strands of DNA as well as does the wild-type protein. The altered protein forms dimers and interacts with T7 DNA polymerase comparable with the wild-type protein. Gene 2.5 protein-R82C condenses single-stranded M13 DNA in a manner similar to wild-type protein when viewed by electron microscopy.
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PMID:A single-stranded DNA-binding protein of bacteriophage T7 defective in DNA annealing. 1274 98

Gene 2.5 of bacteriophage T7 is an essential gene that encodes a single-stranded DNA-binding protein (gp2.5). Previous studies have demonstrated that the acidic carboxyl terminus of the protein is essential and that it mediates multiple protein-protein interactions. A screen for lethal mutations in gene 2.5 uncovered a variety of essential amino acids, among which was a single amino acid substitution, F232L, at the carboxyl-terminal residue. gp2.5-F232L exhibits a 3-fold increase in binding affinity for single-stranded DNA and a slightly lower affinity for T7 DNA polymerase when compared with wild type gp2.5. gp2.5-F232L stimulates the activity of T7 DNA polymerase and, in contrast to wild-type gp2.5, promotes strand displacement DNA synthesis by T7 DNA polymerase. A carboxyl-terminal truncation of gene 2.5 protein, gp2.5-Delta 26C, binds single-stranded DNA 40-fold more tightly than the wild-type protein and cannot physically interact with T7 DNA polymerase. gp2.5-Delta 26C is inhibitory for DNA synthesis catalyzed by T7 DNA polymerase on single-stranded DNA, and it does not stimulate strand displacement DNA synthesis at high concentration. The biochemical and genetic data support a model in which the carboxyl-terminal tail modulates DNA binding and mediates essential interactions with T7 DNA polymerase.
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PMID:The carboxyl-terminal domain of bacteriophage T7 single-stranded DNA-binding protein modulates DNA binding and interaction with T7 DNA polymerase. 1276 55


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