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)

Based on previous in vivo genetic analysis of bacteriophage lambda growth, we have developed two in vitro lambda DNA replication systems composed entirely of purified proteins. One is termed 'grpE-independent' and consists of supercoiled lambda dv plasmid DNA, the lambda O and lambda P proteins, as well as the Escherichia coli dnaK, dnaJ, dnaB, dnaG, ssb, DNA gyrase and DNA polymerase III holoenzyme proteins. The second system includes the E.coli grpE protein and is termed 'grpE-dependent'. Both systems are specific for plasmid molecules carrying the ori lambda DNA initiation site. The major difference in the two systems is that the 'grpE-independent' system requires at least a 10-fold higher level of dnaK protein compared with the grpE-dependent one. The lambda DNA replication process may be divided into several discernible steps, some of which are defined by the isolation of stable intermediates. The first is the formation of a stable ori lambda-lambda O structure. The second is the assembly of a stable ori lambda-lambda O-lambda P-dnaB complex. The addition of dnaJ to this complex also results in an isolatable intermediate. The dnaK, dnaJ and grpE proteins destabilize the lambda P-dnaB interaction, thus liberating dnaB's helicase activity, resulting in unwinding of the DNA template. At this stage, a stable DNA replication intermediate can be isolated, provided that the grpE protein has acted and/or is present. Following this, the dnaG primase enzyme recognizes the single-stranded DNA-dnaB complex and synthesizes RNA primers. Subsequently, the RNA primers are extended into DNA by DNA polymerase III holoenzyme. The proposed model of the molecular series of events taking place at ori lambda is substantiated by the many demonstrable protein-protein interactions among the various participants.
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PMID:Initiation of lambda DNA replication with purified host- and bacteriophage-encoded proteins: the role of the dnaK, dnaJ and grpE heat shock proteins. 252 44

We have established an in vitro system, composed of highly purified bacteriophage lambda and Escherichia coli proteins, that specifically replicates supercoiled templates bearing the lambda replication origin (ori lambda). The complete system is composed of three groups of proteins: the virus-encoded initiator proteins (the lambda O and P proteins), the E. coli replication fork propagation machinery (single-stranded DNA-binding protein, dnaB helicase, dnaG primase, DNA polymerase III holoenzyme, and DNA gyrase), and two bacterial heat shock proteins (dnaJ and dnaK proteins). DNA replication in this system is initiated at or near ori lambda and proceeds unidirectionally rightwards through theta-structure intermediates, ultimately yielding a pair of intertwined daughter circles as the final product. In striking contrast to the situation in vivo and in crude in vitro systems, initiation of lambda DNA replication in the purified protein system does not require "transcriptional activation" of the origin region by E. coli RNA polymerase. We conclude that E. coli primase generates the primers for all leading and lagging strand DNA chains synthesized in this reconstituted lambda replication system.
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PMID:Reconstitution of a nine-protein system that initiates bacteriophage lambda DNA replication. 253 26

Leading and lagging strand DNA synthesis at the replication fork of bacteriophage T7 DNA requires the helicase and primase activities of the gene 4 protein. Gene 4 protein consists of two colinear polypeptides of 56- and 63-kDa molecular mass. We have demonstrated previously that the 56-kDa protein possesses helicase but lacks primase activity (Bernstein, J. A., and Richardson, C. C. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 396-400). The 63-kDa gene 4 protein has now been purified from extracts of T7-infected cells. The preparation contains 5-10% contaminating 56-kDa protein, as shown by Western analysis using polyclonal antibodies to the purified 56-kDa protein. The 63-kDa protein catalyzes DNA-dependent dTTP hydrolysis and has helicase activity; both specific activities are similar to those determined for the 56-kDa protein. The 63-kDa protein efficiently synthesizes sequence-specific di-, tri-, and tetraribonucleotides and stimulates the elongation of tetraribonucleotides by T7 DNA polymerase. Although the 56-kDa protein alone lacks primase activity, it enhances the primase activity of the 63-kDa protein 4-fold. This stimulation can be accounted for by a similar increase in the amount of primers synthesized by the 63-kDa protein in the presence of the 56-kDa protein.
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PMID:Characterization of the helicase and primase activities of the 63-kDa component of the bacteriophage T7 gene 4 protein. 254 45

A new type of radiation-sensitive mutant of S. cerevisiae is described. The recessive radH mutation sensitizes to the lethal effect of UV radiations haploids in the G1 but not in the G2 mitotic phase. Homozygous diploids are as sensitive as G1 haploids. The UV-induced mutagenesis is depressed, while the induction of gene conversion is increased. The mutation is believed to channel the repair of lesions engaged in the mutagenic pathway into a recombination process, successful if the events involve sister-chromatids but lethal if they involve homologous chromosomes. The sequence of the RADH gene reveals that it may code for a DNA helicase, with a Mr of 134 kDa. All the consensus domains of known DNA helicases are present. Besides these consensus regions, strong homologies with the Rep and UvrD helicases of E. coli were found. The RadH putative helicase appears to belong to the set of proteins involved in the error-prone repair mechanism, at least for UV-induced lesions, and could act in coordination with the Rev3 error-prone DNA polymerase.
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PMID:RADH, a gene of Saccharomyces cerevisiae encoding a putative DNA helicase involved in DNA repair. Characteristics of radH mutants and sequence of the gene. 255 5

Ultraviolet light induced pyrimidine dimers in DNA are recognized and repaired by a number of unique cellular surveillance systems. At the highest level of complexity Escherichia coli (E. coli) has a uvr DNA repair system comprising the UvrA, UvrB and UvrC proteins responsible for incision. There are several preincision steps governed by this pathway which includes an ATP-dependent UvrA dimerization reaction required for UvrAB nucleoprotein formation. This complex formation driven by ATP binding, is associated with localized topological unwinding of DNA. This protein complex can catalyze an ATP-dependent 5'----3' directed strand displacement of D-loop DNA or short single strands annealed to a single stranded circular or linear DNA. This putative translocational process is arrested when damaged sites are encountered. The complex is now primed for dual incision catalyzed by UvrC. The remainder of the repair process involves UvrD (helicase II) and DNA polymerase I for a coordinately controlled "excision resynthesis" step accompanied by UvrABC turnover. Furthermore, it is proposed that levels of repair proteins can be regulated by proteolysis. UvrB is converted to truncated UvrB* by a stress induced protease which also acts at similar sites on the E. coli Ada protein. Although UvrB* can bind with UvrA to DNA it cannot participate in helicase or incision reactions. It is also a DNA-dependent ATPase.
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PMID:Dynamics of the Escherichia coli nucleotide excision repair system. 266 5

The T antigen specified by SV40 virus is the only viral-encoded protein required for replication of SV40 DNA. T antigen has two activities that appear to be essential for viral DNA replication: specific binding to duplex DNA at the origin of replication and helicase activity that unwinds the two DNA strands. As judged by electron microscopy, DNA unwinding is initiated at the origin of replication and proceeds bidirectionally. Either linear or circular DNA molecules containing the origin of replication are effective substrates; with closed circular DNA, a topoisomerase capable of removing positive superhelical turns is required for an efficient reaction. Presence of an origin sequence on duplex DNA and a single-strand DNA-binding protein appear to be the only requirements for T antigen to catalyze unwinding. This reaction mediated by T antigen defines a likely pathway to precise initiation of DNA replication: (i) the sequence-specific binding activity locates the origin sequence, (ii) the duplex DNA is unwound at this site, and (iii) the DNA polymerase and primase begin DNA replication. A similar pathway has been inferred for the localized initiation of DNA replication by bacteriophage lambda and by Escherichia coli in which a sequence-specific binding protein locates the origin and directs the DnaB helicase to this site. Observations with the SV40 system indicate that localized initiation of duplex DNA replication may be similar for prokaryotes and eukaryotes.
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PMID:Unwinding of duplex DNA from the SV40 origin of replication by T antigen. 282 89

A DNA replication system was developed that could generate rolling-circle DNA molecules in vitro in amounts that permitted kinetic analyses of the movement of the replication forks. Two artificial primer-template DNA substrates were used to study DNA synthesis catalyzed by the DNA polymerase III holoenzyme in the presence of either the preprimosomal proteins (the primosomal proteins minus the DNA G primase) and the Escherichia coli single-stranded DNA binding protein or the DNA B helicase alone. Helicase activities have recently been demonstrated to be associated with the primosome, a mobile multiprotein priming apparatus that requires seven E. coli proteins (replication factor Y (protein n'), proteins n and n'', and the products of the dnaB, dnaC, dnaG, and dnaT genes) for assembly, and with the DNA B protein. Consistent with a rolling-circle mechanism in which a helicase activity permitted extensive (-) strand DNA synthesis on a (+) single-stranded, circular DNA template, the major DNA products formed were multigenome-length, single-stranded, linear molecules. The replication forks assembled with either the preprimosome or the DNA B helicase moved at the same rate (approximately 730 nucleotides/s) at 30 degrees C and possessed apparent processivities in the range of 50,000-150,000 nucleotides. The single-stranded DNA binding protein was not required to maintain this high rate of movement in the case of leading strand DNA synthesis catalyzed by the DNA polymerase III holoenzyme and the DNA B helicase.
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PMID:The Escherichia coli preprimosome and DNA B helicase can form replication forks that move at the same rate. 282 2

The primosome is a mobile multienzyme DNA replication-priming complex that requires seven Escherichia coli proteins for assembly (the products of the dnaB, dnaC, dnaG, and dnaT genes as well as proteins n and n" and replication factor Y). It has been shown previously that the primosome, in combination with the E. coli DNA polymerase III holoenzyme, can form replication forks in vitro that move at rates similar to those measured in vivo and that the primosome and one of the components of the primosome, the DNA B protein, have DNA helicase activity. Evidence is presented here that another component of the primosome, replication factor Y, possesses DNA helicase activity as well. Factor Y helicase activity requires the presence of E. coli single-stranded DNA binding protein, Mg2+, and hydrolyzable ATP or dATP. Helicase activity is stimulated 15-fold when the enzyme is actively loaded onto single-stranded DNA through a primosome assembly site, and duplex DNA is unwound unidirectionally, 3'----5', along the DNA strand to which the protein is bound.
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PMID:Escherichia coli replication factor Y, a component of the primosome, can act as a DNA helicase. 282 88

Recently, a method has been developed to identify regions in the genome of herpes simplex virus type 1 (HSV-1) which contain genes required for DNA synthesis from an HSV-1 origin of DNA replication, and seven genomic loci have been identified as representing the necessary and sufficient gene set for such replication (C. A. Wu, N. J. Nelson, D. J. McGeoch, and M. D. Challberg, J. Virol. 62:435-443, 1988). Two of the loci represent the well-known genes for DNA polymerase and major DNA-binding protein, but the remainder had little or no previous characterization. In this report we present the DNA sequences of the five newly identified genes and their deduced transcript organizations and encoded amino acid sequences. These genes were designated UL5, UL8, UL9, UL42, and UL52 and were predicted to encode proteins with molecular weights of, respectively, 99,000, 80,000, 94,000, 51,000, and 114,000. All of these genes had clear counterparts in the genome of the related alphaherpesvirus varicella-zoster virus, but only UL5 and UL52 were detectably conserved in the distantly related gammaherpesvirus Epstein-Barr virus, as judged by amino acid sequence similarity. The sequence of the UL5 protein, and of its counterparts in the other viruses, contained a region closely resembling known ATP-binding sites; this could be indicative, for instance, of a helicase or primase activity.
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PMID:Structures of herpes simplex virus type 1 genes required for replication of virus DNA. 282 7

The dnaB gene of Escherichia coli encodes a helicase that operates at replication forks of the bacterium and certain of its bacteriophages to produce separated strands suitable for subsequent use by primase and DNA polymerase III. Here, we present the sequence of the dnaB gene of Salmonella typhimurium, a functionally interchangeable analog of the E. coli dnaB gene. The DnaB proteins of these two organisms, inferred from the DNA sequences, are identical in length and in 93% of amino acid residues. Extended portions of the DnaB proteins are also similar to two phage-encoded DNA replication proteins: the gene 4 helicase-primase of coliphage T7 and, as reported previously (H. Backhaus and J. B. Petri, Gene 32: 289-303, 1984), the gene 12 protein of Salmonella phage P22. In contrast, little similarity was found between DnaB and either the UvrD repair helicase or transcription termination factor Rho (an RNA-DNA helicase). These results identify S. typhimurium DnaB as a member of the DnaB family of proteins by structural, as well as functional, criteria and provide the basis for the eventual identification, by mutational studies, of residues in DnaB critical for its function.
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PMID:Sequence of the dnaB gene of Salmonella typhimurium. 283 67


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