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:3.1.26.4 (RNase H)
2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A specific cleavage by the reverse transcriptase-associated RNase H activity generates the RNA primer for plus strand DNA synthesis during reverse transcription. Previously, we used site-directed mutagenesis to define the sequence features of the polypurine tract (PPT) required for correct plus strand priming by the Moloney murine leukemia virus (M-MuLV) reverse transcriptase (Rattray, A. J., and Champoux, J. J. (1989) J. Mol. Biol. 208, 445-456). Although the sequences of human immunodeficiency virus type 1 (HIV-1) and M-MuLV diverge completely outside a 20-base region encompassing the PPT, within this region there are only three differences between the two viruses. Here we show that the HIV-1 reverse transcriptase will utilize the M-MuLV PPT as an origin for plus strand initiation in vitro. This finding enabled us to use the set of PPT mutants previously generated in M-MuLV, in conjunction with a small set of newly derived mutations within the HIV-1 PPT, to study plus strand priming by the HIV-1 reverse transcriptase. Despite the similarity between the two PPT regions, the sequence features important for positioning RNase H for the cleavage reaction that generates the plus strand primer are different for the two viruses. For M-MuLV, the -7A residue is a critical specificity determinant in the priming reaction, whereas for HIV-1, the -2G and -4G residues play key roles in determining the specificity of priming.
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PMID:The sequence features important for plus strand priming by human immunodeficiency virus type 1 reverse transcriptase. 768 Oct 62

Illimaquinone, a natural marine product, was shown by us to inhibit preferentially the ribonuclease H (RNase H) activity of the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). We have also shown that illimaquinone inhibits the RNase H activity of HIV-2 RT in addition to that of HIV-1 RT, murine leukemia virus RT, and Escherichia coli RNase H. Chemical modifications of HIV-1 RT by sulfhydryl-specific reagents, such as N-ethylmaleimide (NEM) have been demonstrated to specifically inhibit the RNase H activity of the enzyme. Since our previous studies have suggested that cysteine 280 in HIV-1 RT interacts with the sulfhydryl reagents, we have examined the possibility that illimaquinone interacts with the RT molecules via amino acid residues located in the vicinity of cysteine 280 in both HIV-1 and HIV-2 RTs. In the combined effect studies of illimaquinone and NEM, the two structurally unrelated compounds were shown to be mutually exclusive, exhibiting an antagonistic interaction with both HIV-1 and murine leukemia virus-associated RNase H activities. This implicates cysteine 280, in both HIV-1 and HIV-2 RTs, to be in close proximity to the putative binding site of the enzyme to illimaquinone. The above conclusion is further supported by the fact that the RNase H activity of an enzymatically active mutant of HIV-1 RT, in which cysteine 280 was replaced by serine, was substantially more resistant to illimaquinone than the corresponding activity of the wild-type enzyme. The fact that NEM failed to inhibit E. coli RNase H as opposed to illimaquinone highlights a major difference between the retroviral and bacterial RNase H.
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PMID:The interaction of illimaquinone, a selective inhibitor of the RNase H activity, with the reverse transcriptases of human immunodeficiency and murine leukemia retroviruses. 768 48

The functional relationship between the polymerase and RNase H domains of reverse transcriptase (RT) was investigated by studying the activities of AKR murine leukemia virus (MuLV) enzymes. In addition to the wild type, an RNase H-minus RT missing the entire RNase H domain and two other mutants having abnormal polymerase:RNase H ratios were expressed. These mutants include (i) a chimeric protein in which the MuLV RNase H domain was replaced by the entire Escherichia coli RNase H sequence and (ii) an RT with a 126 amino acid deletion in a region analogous to the "connection" subdomain in the p66 subunit of human immunodeficiency virus type 1 RT (Kohlstaedt, L. A., Wang, J., Friedman, J. M., Rice, P. A., & Steitz, T. A. (1992) Science 256, 1783-1790). With the wild-type RT, the major RNase H cleavage reaction was coordinated with DNA synthesis and occurred at a position corresponding to 15 nucleotides from the 3'-terminus of the DNA primer. Additional cleavages closer to the 5'-end of the RNA were explained in terms of a model relating binding of the RNA.DNA hybrid substrate and enzyme structure. The chimeric RT behaved like E. coli RNase H, exhibited 300-fold higher RNase H activity than wild-type RT, and was limited in its ability to synthesize DNA. Qualitative and quantitative changes in the polymerase and RNase H activities of the deletion mutant were also observed. The RNase H domain appeared to function independently of the polymerase domain, supporting the idea that the proper spatial relationship between the two active centers was disrupted by the mutation. Taken together, our results indicate that alteration of the normal polymerase:RNase H ratio can have profound effects on both polymerase and RNase H cleavage activities, as expected for an enzyme with two interdependent domains.
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PMID:A large deletion in the connection subdomain of murine leukemia virus reverse transcriptase or replacement of the RNase H domain with Escherichia coli RNase H results in altered polymerase and RNase H activities. 768 24

The quinoline U-78036 represents a new class of non-nucleoside human immunodeficiency virus (HIV)-1 reverse transcriptase inhibitors. The agent possesses excellent antiviral activity at nontoxic doses in HIV-1-infected lymphocytes grown in tissue culture. Enzymatic kinetic studies of the HIV-1 reverse transcriptase (RT)-catalyzed RNA-directed DNA polymerase function were carried out in order to determine whether the inhibitor interacts with the template-primer or deoxyribonucleotide triphosphate (dNTP) binding sites of the polymerase. The data were analyzed using steady-state or Briggs-Haldane kinetics assuming that the template-primer binds to the enzyme first followed by the dNTP and that the polymerase functions processively. The calculated rate constants are in agreement with this model. The results show that the inhibitor acts as a mixed to noncompetitive inhibitor with respect to both the template-primer and the dNTP binding sites of the enzyme. Hence, U-78036 inhibits the RNA-directed DNA polymerase activity of RT by interacting with a site distinct from the template-primer and dNTP binding sites. Moreover, the potency of U-78036 is dependent on the base composition of the template-primer. The equilibrium constants for various enzyme-substrate-inhibitor complexes were at least seven times lower for the poly(rC).(dG)10-catalyzed system than the one catalyzed by poly(rA).(dT)10. In addition, the inhibitor does not impair the DNA-dependent DNA polymerase activity and the RNase H function of HIV-1 RT nor does it inhibit the RNA-directed DNA polymerase activity of the HIV-2, avian myoblastoma virus, and murine leukemia virus RT enzymes.
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PMID:The quinoline U-78036 is a potent inhibitor of HIV-1 reverse transcriptase. 768 7

RNA/DNA substrates derived from the 5' ends of human immunodeficiency virus (HIV) and Moloney murine leukemia virus (MMuLV) genomes were used to study the specificity of the RNase H activities of HIV, AMV (avian myeloblastosis virus), and MMuLV reverse transcriptases. These substrates were selected because they represent the site for the first template switch during proviral DNA synthesis. Variability of cleavage was observed depending on the origin of the enzyme as well as the sequence of the RNA/DNA substrate. The minimal size of hybrid recognized by the RNase H activity of reverse transcriptase was also affected by the same parameters, namely, the enzyme and the substrate origin. Moreover, the size of the residual 5'-undigested RNA after completion of the RNase H reaction depended on the position of the DNA annealed to the genomic RNA. When the hybrid was located at the 5' R region of the viral genome, stable hybrids with RNAs of 13-18 nucleotides remained following digestion by HIV reverse transcriptase, and 21-24 nucleotides following digestion by AMV reverse transcriptase and MMuLV reverse transcriptase. On the other hand, with all three enzymes, smaller sized hybrids remained when the DNA was hybridized to internal U5 or R sequences. The reason for this variance in size appears to be the inability of RNase H to efficiently digest at the 5' end of hybrid structures. Surprisingly, hybridization to the RNA template, of a DNA oligomer that extended 15 nucleotides beyond the 5' end of the RNA R region sequences, resulted in further digestion of the RNA. This unexpected mode of action of RNase H at the 5' end of the genomic RNA should be taken in consideration in studies of the first template switch.
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PMID:RNase H activity of reverse transcriptases on substrates derived from the 5' end of retroviral genome. 768 65

We have examined the ability of reverse transcriptases (RT) to catalyze strand transfer from internal regions of RNA templates, resulting in switching of a primer from one template to another. To study this phenomenon, we employed a system of donor and acceptor templates in which homologous strand transfer can occur from a homopolymeric sequence, positioned internally on the donor template. Our results indicate that reverse transcriptases from human immunodeficiency virus (HIV), avian myeloblastosis virus (AMV), and murine leukemia virus (MuLV) are all able to catalyze strand transfer from this sequence. Catalysis of this reaction is not dependent upon ribonuclease H (RNase H) activity, since an RNase H-deficient form of HIV-RT is able to catalyze the reaction efficiently. Additionally, N-ethylmaleimide, which inhibits RNase H but not polymerase activity, did not inhibit the template switching by either the native or RNase H-deficient forms of HIV-RT. Our data further indicate that template switching may be promoted by RT pausing at a specific site on the donor template. Conditions that increase RT pausing at this site also increase template switching. These results suggest that transient RT pausing at specific sites on the viral genome during reverse transcription may promote template switches that in turn lead to recombination.
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PMID:Pausing by retroviral DNA polymerases promotes strand transfer from internal regions of RNA donor templates to homopolymeric acceptor templates. 769 74

RNases H are traditionally thought to degrade RNA only in RNA-DNA hybrid form. We found that the wild-type Moloney murine leukemia virus (M-MuLV) reverse transcriptase (RT) was capable of degrading RNA in RNA-RNA duplexes as well as in RNA-DNA hybrids, as assayed by in situ gel techniques. Escherichia coli RNase H does not degrade the RNA-RNA duplex in this assay, while E. coli RNase III, a double-strand-specific ribonuclease, does. The apparent specific activity of M-MuLV RT on RNA-RNA duplexes is similar to that on RNA-DNA hybrids. Neither the DNA polymerase domain nor the RNase H domain of RT expressed individually exhibited this RNA-RNA activity. We have generated a series of mutations in the RNase H domain of M-MuLV RT, expressed the mutant enzymes in E. coli, and assayed these mutants for various activities. All RTs were as active as the wild type in the oligo(dT):poly(rA) DNA polymerase assay, and many retained both nuclease activities. Two enzymes with mutations at the carboxyl terminus of the RNase H domain retained RNA-DNA activity, but not RNA-RNA activity. Another mutant enzyme showed the opposite phenotype, retaining RNA-RNA, but not RNA-DNA, nuclease activity. Thus, we were able to genetically separate the two activities. These results may be helpful in defining enzyme-substrate interactions.
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PMID:Nuclease activities of Moloney murine leukemia virus reverse transcriptase. Mutants with altered substrate specificities. 769 92

We have previously demonstrated, in vitro, that phosphodiester and phosphorothioate antisense oligodeoxynucleotides could direct ribonuclease H to cleave non-target RNA sites and that chimeric methylphosphonodiester/phosphodiester analogue structures were substantially more specific. In this report we show that such chimeric molecules can promote point mutation-specific scission of target mRNA by both Escherichia coli and human RNases H in vitro. Intact human leukaemia cells 'biochemically microinjected' with antisense effectors demonstrated efficient suppression of target mRNA expression. It was noted that the chimeric methylphosphonodiester/phosphodiester structures showed single base discrimination, whereas neither the phosphodiester nor phosphorothioate compounds were as stringent. Finally, we show that the antisense effects obtained in intact cells were due to endogenous RNase H activity.
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PMID:Single base discrimination for ribonuclease H-dependent antisense effects within intact human leukaemia cells. 773 9

Poly(A) RNA was isolated from microdissected guinea pig crista ampullaris epithelium and converted into cDNA with RNase H- murine leukemia virus reverse transcriptase. After size fractionation, the cDNA was directionally ligated into the vector pSPORT 1 and the plasmids electroporated into E. coli. The library was found to have 1.6 x 10(7) independent colonies with 5% of the colonies lacking an insert. Thirty randomly selected colonies were checked for inserts and the average insert size was 833 base pairs with a range of 400 to 2300 base pairs. The library was screened with a beta-actin guinea pig cDNA probe and 0.16% of the colonies contained an insert hybridizing to the probe.
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PMID:Construction of a cDNA library from microdissected guinea pig crista ampullaris. 815 7

We have previously described the in vitro and in vivo characterization of a panel of mutations affecting the RNase H domain of Moloney murine leukemia virus reverse transcriptase (Blain, S. W., and Goff, S.P. (1993) J. Biol. Chem. 268, 23585-23592; Blain, S. W., and Goff, S. P. (1995) J. Virol. 69, 4440-4452). We were intrigued by a discrepancy between in vitro and in vivo RNase H results for two of the mutants. While delta C and delta 5E appeared to have nearly wild-type RNase H activity in vitro, they were unable to degrade their genomic RNA in vivo and thus were effectively RNase H null mutants in this context. In this present report, we describe the differential effects of these mutations on RNase H activity in vitro in the presence of Mg2+ versus Mn2+: mutants delta C and delta 5E were active in the presence of the less biologically relevant Mn2+ and not in the presence of Mg2+. We also describe three mutants with only partial activity in Mg2+. The presence of the different cations can also affect DNA polymerization and processivity of an RNase H-deficient mutant.
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PMID:Differential effects of Moloney murine leukemia virus reverse transcriptase mutations on RNase H activity in Mg2+ and Mn2+. 857 37


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