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:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

HIV-IN protein, tagged with a hexahistidine tail was expressed in Escherichia coli and purified by a one-step nickel chelate affinity chromatography procedure. The purified IN protein was characterized in terms of its endonuclease and integrase properties in vitro. Specific cleavage and integration of HIV U5 LTR ends were observed in the presence of 2-5 mM Mg2+ or Ca2+. In the presence of 2 mM Mn2+, cleavage and integration occurred at additional sites indicating a decreased specificity. The properties of mutant IN proteins were examined in vitro. Deletion of 39 amino acids from the N-terminus and a minimum of 25 residues from the C-terminus impaired IN-mediated cleavage and integration activities. The results of site-directed mutagenesis experiments showed that residues critical for IN function are highly conserved. Mutations of conserved residues Asp64, Pro109, Asp116, and Glu152 adversely affected IN function in vitro. Mutations of nonconserved residues Gly189 and Thr112 had no effect. Mutation of a conserved Thr115 to Ala caused a near complete loss of Mg(2+)-dependent integration activity, but only partially effected endonucleolytic cleavage activity of IN. These results suggest that not all conserved residues are involved in both endonucleolytic cleavage and integration activities of HIV-IN.
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PMID:Identification of amino acid residues critical for endonuclease and integration activities of HIV-1 IN protein in vitro. 158 29

Five cassettes of the pol gene of human immunodeficiency virus 1 were constructed and inserted under the control of the polyhedrin gene promoter of Autographa californica nuclear polyhedrosis virus by homologous recombination. The first cassette polF contains the full-length pol open reading frame; the second cassette pol100 starts with the first AUG codon of the pol gene and deletes 103 amino acids from the amino terminus of the pol gene product; the third cassette pol97 deletes the entire protease coding sequence; the fourth cassette pol66 deletes both the protease and endonuclease/integrase coding sequences; and the fifth cassette pol51 contains the reverse transcriptase coding sequences plus 39 3'-terminal nucleotides of the RNase H coding sequences. We have expressed these five forms of the pol gene in Spodoptera frugiperda SF9 cells and have analyzed for both reverse transcriptase and RNase H activities. The polF construct expressed several processed forms, 66 kDa, 51 kDa, and 34 kDa proteins, that were detected only by Western blot. In contrast, pol100, pol97, pol66, and pol51 products were expressed at high levels and were readily detectable in gels by staining. The levels of expression of these four products were estimated to be greater than 150 mg/liter of culture (5 x 10(8) cells). Activity gel analyses showed that the pol100, pol97, pol66, and pol51 products possess reverse transcriptase activity; however, only pol97 and pol66 have RNase H activity. Our results demonstrate that many forms, including partially cleaved forms of human immunodeficiency virus 1 pol gene products, possess reverse transcriptase activity but only certain forms have RNase H activity.
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PMID:Enzyme activities in four different forms of human immunodeficiency virus 1 pol gene products. 171 Dec 3

The cIII gene product of lambdoid bacteriophages promotes lysogeny by stabilizing the phage-encoded CII protein, a transcriptional activator of the repressor and integrase genes. Previous works showed that the synthesis of the bacteriophage lambda CIII protein has specific translational requirements imposed by the structure of the mRNA. To gain insight into the mRNA structure and its role in regulating cIII translation, we undertook a mutational analysis of the cIII gene of the related bacteriophage HK022. Our data support the hypothesis that in HK022, as in lambda, translation initiation requires a specific mRNA structure. In addition, we found that translation of HK022 cIII, like that of lambda, is strongly reduced in a host deficient in the endonuclease RNase III.
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PMID:Genetic analysis of the cIII gene of bacteriophage HK022. 182 68

A quantitative and efficient assay was developed to measure the 3'-OH terminal DNA endonuclease activity of the avian myeloblastosis virus (AMV) integrase protein. A retroviral-like linearized plasmid containing long terminal repeat (LTR) sequences at its recessed 3'-OH termini was filled in and labeled with the Escherichia coli Klenow DNA polymerase fragment. The 32P-labeled nucleotide was located at the penultimate position. The labeled linearized plasmid or restriction fragments derived from it were incubated with AMV IN and release of the label was quantitated by conversion to acid-soluble counts. The structure of the released product was characterized on 23% sequencing gels. Results indicate that AMV integration protein is functioning as an endonuclease releasing a dinucleotide and that the activity is stoichiometric with a preference for the cleavage of the U3 LTR terminus over that of the U5 LTR terminus.
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PMID:Development of an acid-soluble assay for measuring retrovirus integrase 3'-OH terminal nuclease activity. 188 32

Recombinant human immunodeficiency virus type 1 (HIV-1) integrase (IN) produced in Escherichia coli efficiently cleaves two nucleotides from the 3' end of synthetic oligonucleotide substrates which mimic the termini of HIV-1 proviral DNA. Efficient cleavage was restricted to HIV-1 substrates and did not occur with substrates derived from other retroviruses. Mutagenesis of the U5 long terminal repeat (LTR) terminus revealed only moderate effects of mutations outside the terminal four bases of the U5 LTR and highlighted the critical nature of the conserved CA dinucleotide motif shared by all retroviral termini. Integration of the endonuclease cleavage products occurs subsequent to cleavage, and evidence that the cleavage and integration reactions may be uncoupled is presented. Competition cleavage reactions demonstrated that IN-mediated processing of an LTR substrate could be inhibited by competition with LTR and non-LTR oligonucleotides.
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PMID:Substrate specificity of recombinant human immunodeficiency virus integrase protein. 189 9

Members of the IS3 family of insertion sequences are found in a wide range of bacteria. At least 10 members of this family carry two major open reading frames: a small upstream frame (0 phase), and a longer downstream frame in the -1 phase. The downstream frame shows significant similarity at the amino acid level. A highly conserved region of this frame also exhibits notable similarity with a region of the integrase (endonuclease) domain of retroviruses. Although the overall transposition mechanism of the insertion sequence and retroviral elements is certainly different, the two groups may share additional common features, including a -1 frameshift resulting in the production of a fusion protein.
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PMID:Functional similarities between retroviruses and the IS3 family of bacterial insertion sequences? 196 20

Human endogenous retroviral element S71 had previously been shown to contain gag- and pol-related regions and a 3' LTR-like sequence. The nucleotide sequence of S71 was determined and compared with the corresponding regions of SSV and its helper virus SSAV. The 1.48-kb S71 gag region consists of matrix protein p15 (MA)-, capsid protein p30 (CA)-, and nucleocapsid protein p10 (NC)-related sections and the 1.82-kb pol region of tether, RNase H (RH), and endonuclease/integrase (IN) sections. The S71 nucleotide sequence contains a 167 amino acid open reading frame encompassing MA. The boundaries of the S71 element are delimited by direct repeats and the entire element is 5.4 kb long. Similarity between S71 and the v-sis-bearing, defective SSV provirus also covers overall structural organization, including the presence of presumably nonretroviral sequences. Both the gag and the pol regions of S71 contain sequences highly conserved in numerous retroviruses. Phylogenetic analysis with conserved CA, RH, and IN sequences showed that of all other (C-type) human retroviral elements available for comparison, S71 is most closely related to infectious primate and murine retroviruses. This suggests that S71 represents a phylogenetic subgroup of its own. In addition we identified short ranges of conserved amino acid sequences within C-type retroviral gag and pol genes sufficient for phylogenetic analysis. Use of these may facilitate large-scale phylogenetic evaluation of C-type retroviral elements and allow rapid classification of new elements.
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PMID:S71 is a phylogenetically distinct human endogenous retroviral element with structural and sequence homology to simian sarcoma virus (SSV). 215 93

A common feature in the life cycle of cytocidal retroviruses, including human immunodeficiency virus type 1 (HIV-1), is the accumulation of large amounts of unintegrated viral DNA. As yet, the role of unintegrated viral DNA in the cytopathogenesis of cytocidal retrovirus infections remains unresolved. HIV-1 mutants which were deleted in the integrase/endonuclease gene and which were unable to establish an integrated form of the virus were constructed. Despite an inability to integrate, these mutants were fully competent templates for HIV-1 core and envelope antigen production. HIV-1 antigen could be detected in the supernatants of lymphocyte cultures infected with HIV-1 integrase mutants. However, an inability to rescue infectious virus from these cultures indicated that HIV-1 integration was required for the production of infectious HIV-1. On the basis of the ability of unintegrated HIV-1 DNA to serve as a template for HIV-1 antigen production, it is plausible that unintegrated viral DNA can contribute to the HIV-1 antigen pool during HIV-1 replication.
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PMID:Integration is not necessary for expression of human immunodeficiency virus type 1 protein products. 215 98

We have determined the complete 9202 nucleotide sequence of the visna lentivirus. The deduced genetic organization most closely resembles that of the AIDS retrovirus in that there is a novel central region separating pol and env. Moreover, there is a close phylogenetic relationship between the conserved reverse transcriptase and endonuclease/integrase domains of the visna and AIDS viruses. These findings support the inclusion of the AIDS virus in the retroviral subfamily Lentivirinae.
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PMID:Nucleotide sequence of the visna lentivirus: relationship to the AIDS virus. 241 Jan 40

A category of viruses has been identified which is related to human immunodeficiency virus (HIV) but is more closely related to a group of simian retroviruses (STLV-III). These viruses named HTLV-IV, LAV-II, or SBL-6669, are prevalent in West-Africa. In this study, we analysed the cross-reactivity at the protein level between HTLV-IV and HIV (HTLV-IIIB). The results indicate that most people infected with HTLV-IV have antibodies that react to the major gag protein of HIV p 24. There is also a high degree of immunologic cross-reactivity between the pol gene products of HIV and HTLV-IV. Among these the endonuclease/integrase is more conserved than the reverse transcriptase. In contrast, the envelope glycoproteins that are the most frequently detected antigens by antibodies from exposed individuals are serotype specific. These data make the env gene products the most interesting antigens for serotype specific diagnosis of human retroviruses infections.
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PMID:A STLV-III related human retrovirus, HTLV-IV: analysis of cross-reactivity with the human immunodeficiency virus (HIV). 244 14


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