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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)

Intact primer RNA for discontinuous DNA replication of Escherichia coli has been detected by specific labeling in vitro of its 5'-terminal tri- (or di-) phosphate group with vaccinia guanylyltransferase and [alpha-32P]GTP. A mutant defective either in RNase H or in both RNase H and DNA polymerase I accumulated about 10 or 30 times more intact primer RNA, respectively, than wild-type cells. The primers started with purine in an A to G ratio of 5 and the most abundant 5'-terminal dinucleotide sequence was (p)ppA-Pu. The chain length of the intact primer RNA was approximately 10 to 12 nucleotide residues. The structural properties of the E. coli primer RNa resemble those of the eukaryotic primer RNA.
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PMID:Evidence that discontinuous DNA replication in Escherichia coli is primed by approximately 10 to 12 residues of RNA starting with a purine. 241 35

The reverse transcriptase polymerase of the human T-cell lymphotropic virus/lymphadenopathy-associated virus has been cloned into an expression vector and expressed in Escherichia coli. Two polypeptides of 66 and 51 kDa molecular mass are detectable in polymerase-expressing bacterial lysates with human patient sera. They are processed from a short-lived 120-kDa polyprotein precursor equivalent to a region consisting of polymerase, protease, and endonuclease. The 51 kDa protein appears to originate from the 66-kDa molecule; additional processing products are 32- and 15-kDa proteins. The bacterially expressed polymerase is enzymatically active and exhibits the template specificities, ion requirements, and response to inhibitors of the authentic enzyme. It was purified by DEAE-cellulose-, phosphocellulose-, and poly(rC)-agarose column chromatography followed by glycerol density gradient centrifugation. It copurifies with an RNase H activity, suggesting the existence of a virus-coded DNA polymerase-RNase H complex. The purified bacterial enzyme allows a safe large-scale screening for inhibitors of both activities.
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PMID:RNase H activity associated with bacterially expressed reverse transcriptase of human T-cell lymphotropic virus III/lymphadenopathy-associated virus. 244 62

We have developed a fast and efficient procedure for generating cDNA libraries in plasmid or phage lambda vectors. We used Mo-MuLV reverse transcriptase to synthesize the first strand and directly added Escherichia coli DNA polymerase I with RNase H to synthesize the second strand. A special advantage of our procedure is the use of oligodeoxynucleotide adapters to insert the cDNA into the vector, avoiding the use of methylating enzymes and subsequent digestion with massive amounts of restriction endonucleases. This also obviates the need to tail the cDNA molecules with homopolymers, simplifying subsequent procedures such as sequencing or transfer to other vectors. Finally, we have used a rapid screening procedure to isolate full-length clones with oligodeoxynucleotide probes recognizing conserved regions at the 5' termini of the mRNA. The system is ideal for cloning and analyzing polymorphic alleles of genes, such as those of the major histocompatibility complex.
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PMID:A rapid and improved method for generating cDNA libraries in plasmid and phage lambda vectors. 244 32

Retroviral reverse transcriptase possesses DNA polymerase and ribonuclease H (RNase H) activity within a single polypeptide. Chemical or proteolytic treatment of reverse transcriptase has been used in the past to produce enzyme that is missing DNA polymerase activity and retains RNase H activity. It has not been possible to obtain reverse transcriptase that lacks RNase H but retains DNA polymerase activity. We have constructed a novel deletion derivative of the cloned Moloney murine leukemia virus (M-MLV) reverse transcriptase gene, expressed the gene in E. coli, and purified the protein to near homogeneity. The purified enzyme has a fully active DNA polymerase, but has no detectable RNase H activity. These results are consistent with, but do not prove, the conclusion that the DNA polymerase and RNase H activities of M-MLV reverse transcriptase reside within separate structural domains.
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PMID:Isolation of cloned Moloney murine leukemia virus reverse transcriptase lacking ribonuclease H activity. 244 47

The reverse transcriptase of Moloney murine leukemia virus, like that of all retroviruses, exhibits a DNA polymerase activity capable of synthesis on RNA or DNA templates and an RNase H activity with specificity for RNA in the form of an RNA.DNA hybrid. We have generated a library of linker insertion mutants of the Moloney murine leukemia virus enzyme expressed in bacteria and assayed these mutants for both enzymatic activities. Those mutations affecting the DNA polymerase activity were clustered in the 5'-proximal two-thirds of the gene, and those affecting RNase H were in the remaining 3' one-third. Based on these maps, plasmids were made that expressed each one of the domains separately; assays of the proteins encoded by these plasmids showed that each domain exhibited only the expected activity.
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PMID:Domain structure of the Moloney murine leukemia virus reverse transcriptase: mutational analysis and separate expression of the DNA polymerase and RNase H activities. 245 Mar 47

Poly(A)+RNA and cytoplasmic RNA of Ehrlich ascites tumor cells grown in vivo were used to study the quality and efficiency of cDNA synthesis. It was found that the rates of oligo(dT)-primed and unprimed reverse transcription were very similar in both cases. The size distributions of the cDNA strands prepared from unfractionated RNA reflected the size of cytoplasmic mRNA populations including a significant fraction of long molecules up to 6 kb. The fraction of cDNAs primed on rRNAs by oligo(dT) was found to be as low as 2-3%. Following second-strand synthesis by means of RNase H-induced nick translation by DNA polymerase I the overall yields in double-stranded cDNA were slightly higher when unfractionated cytoplasmic RNA was used as starting template. In repeated experiments we obtained an average yield of 2.2 micrograms of double-stranded cDNA when 70 micrograms of unfractionated cytoplasmic RNA was used as starting material. This amount of cDNA synthesized in one assay was sufficient to construct representative cDNA libraries in different vectors. Southern hybridizations of DNA isolated from cDNA libraries with various radiolabelled probes show that the libraries constructed from cDNA synthesized from cytoplasmic RNA not enriched in poly(A)+RNA contain a high ratio of full-length cDNA clones. The results suggest that representative cDNA libraries of high quality can be constructed without pre-isolation of poly(A)+RNA fractions.
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PMID:Construction and quality of cDNA libraries prepared from cytoplasmic RNA not enriched in poly(A)+RNA. 246 57

A plasmid construct expressing the p66 version of the human immunodeficiency virus reverse transcriptase as a bacterial fusion protein was subjected to in vitro mutagenesis, and the resulting variant proteins were assayed to define the locations of the two major enzymatic activities. The DNA polymerase activity was localized to the N-terminal portion of the protein; mutations altering or eliminating the C-terminal portion had little or no effect on that activity. The results suggest that, in contrast with previous reports, the p51 subunit found in virions should exhibit DNA polymerase activity. Mutations in many parts of the protein eliminated RNase H activity, suggesting that several areas are needed for proper folding and generation of that activity.
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PMID:Linker insertion mutagenesis of the human immunodeficiency virus reverse transcriptase expressed in bacteria: definition of the minimal polymerase domain. 247 90

Mechanisms that could operate to initiate pBR322 DNA replication in the absence of RNase H and DNA polymerase I are described. Two different pathways leading to extensive unwinding of pBR322 DNA have been observed under DNA replication reaction conditions in vitro. In the presence of RNA polymerase and DNA gyrase, specifically initiated RNA II (the leading-strand primer precursor) can form an RNA-DNA hybrid with the template that starts just upstream of the origin of DNA replication and continues for about 3 kilobases. Subsequent digestion of the RNA in this RNA-pBR322 DNA hybrid results in the formation of a highly unwound DNA termed form I. If DNA gyrase is absent during the RNA polymerase-catalyzed elongation of RNA II, a stable RNA-pBR322 DNA hybrid can still form that is localized to the origin region of the genome. Formation of this hybrid activates the primosome assembly site present on the lagging-strand DNA template, by displacing it to a single-stranded conformation, thereby allowing preprimosome assembly. Once assembled, the DNA helicase activity of the preprimosome, in the presence of the single-stranded DNA binding protein and DNA gyrase but in the absence of any further transcription, can also result in extensive unwinding of pBR322 DNA. The product of this reaction, form I DNA, is more unwound than form I DNA. The formation of both form I and form I DNA is inhibited by the presence of excess RNA I, as well as by RNase H at concentrations sufficient to catalyze the normal processing of RNA II required for initiation of leading-strand DNA synthesis. These results suggest that RNA II-pBR322 DNA hybrid formation is essential to permit preprimosome assembly during pBR322 DNA replication under conditions where both RNase H and DNA polymerase I are absent.
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PMID:Transcriptional activation of pBR322 DNA can lead to duplex DNA unwinding catalyzed by the Escherichia coli preprimosome. 247 95

We have developed a modified primer extension procedure for specific detection of mRNA. Alkali-fragmented total cellular RNA or some RNA fraction is hybridized to single-stranded or double-stranded M13 DNA containing the insert of interest which is immobilized on nylon membranes. Hybridized RNA is then detected by incubation of membranes with Escherichia coli RNase H and DNA polymerase I. RNase H is used for nicking the RNA in the hybrids. The resulting 3'-OH groups can subsequently be used by DNA polymerase I to synthesize a labeled complementary strand. The method described is both relatively fast and sensitive and particularly useful for screening large numbers of DNA clones for their representation in RNA populations. Using total cellular RNA as hybridization probe and single-stranded M13 DNA as template as low as 0.25 ng of a specific mRNA was detected (2.5-fold background) when adding 1 microCi [3H]dCTP or 2.5 microCi [32P]d-CTP alternatively as radioactive precursor for the labeling reaction. The detection limit increased to 1 ng (2-fold background) with denatured replicative form double-stranded M13 DNA as template.
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PMID:A modified primer extension procedure for specific detection of DNA-RNA hybrids on nylon membranes. 247 44

In Escherichia coli the dnaQ+ gene, which encodes epsilon, a fidelity subunit of DNA polymerase III, and the rnh+ gene, which encodes RNase H, share a promoter region but are transcribed in opposite directions. The presence of this divergent transcriptional unit on a multicopy plasmid inhibited by as much as 10-fold mutations induced by the SOS-dependent mutagens methyl methanesulfonate and UV light. Mutations in either gene eliminated the effect, suggesting that both genes contribute either directly or indirectly to the antimutagenic phenotype. Neither survival to mutagen exposure nor induction of the SOS response was comparably affected by the presence of the genes. Although the antimutagenic phenotype was partially suppressed by excess UmuDC proteins, which are required for SOS mutagenesis, the presence of the dnaQ+-rnh+ clone also reduced the induction of mutations by N-methyl-N'-nitro-N-nitrosoguanidine in cells deficient for SOS mutagenic processing. The results suggest that the presence of the dnaQ+-rnh+ divergent transcriptional unit interferes with an underlying mutagenic mechanism that is normally facilitated by the proteins induced as part of the SOS response.
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PMID:Presence of the dnaQ-rnh divergent transcriptional unit on a multicopy plasmid inhibits induced mutagenesis in Escherichia coli. 254 18


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