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)

Ultraviolet thermal denaturation studies substantiate our earlier hypothesis that substitution of a L-nucleotide residue for a D-nucleotide within a DNA duplex permits a stable structure in which all bases are paired through Watson-Crick hydrogen bonds (Damha, M. J., Giannaris, P. A., Marfey, P., & Reid, L. S. (1991) Tetrahedron Lett. 32, 2573-2576). This conclusion is also evident from the NMR work of Blommers et al. [Blommers, M. J. J., et al. (1994) Biochemistry (following paper in this issue)]. Our thermal denaturation studies indicate that, while weakening the interaction with target DNA and RNA, these substitutions allow for excellent cooperative binding. When the target is single-stranded DNA, the melting temperature of the complex is lowered by 4-5 degrees C per L-dU incorporation and by 0.4-2.6 degrees C when an internal D-dC is replaced by L-dC (1 M NaCl). When the target is RNA, the depression of Tm is also greater for L-dU substitutions (5-8 degrees C) than for L-dC substitutions (2-4 degrees C). The depressions of Tm caused by introducing A/C and G/T mismatches at the same positions were significantly greater. L/D-DNA chimeras were found to activate RNase H cleavage when hybridized to RNA. Furthermore, the stability of chimeric L/D-DNA against degradation by various commercial phosphodiesterases was found to be significant, as was their stability against digestion in human serum. These experiments establish that L/D-DNA chimeras serve as excellent models of antisense oligonucleotides.
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PMID:Antisense L/D-oligodeoxynucleotide chimeras: nuclease stability, base-pairing properties, and activity at directing ribonuclease H. 801 50

Okazaki fragments are important intermediates in DNA replication. Chimeric duplexes that are structurally equivalent to Okazaki fragments also occur during reverse transcription of RNA retroviruses. Such duplexes consist of an RNA-DNA chimeric strand base-paired to a pure DNA strand; hence they have a hybrid duplex "left half" covalently linked to a "right half" that is pure DNA. We have determined the solution structure of the synthetic Okazaki fragment r(gcg)d(TATACCC):d(GGGTATACGC) by means of two-dimensional NMR, restrained molecular dynamics and full relaxation matrix simulation of the two-dimensional nuclear Overhauser effect spectra at various mixing times. The large negative x-displacement and large positive inclination in the hybrid section of the duplex are structural characteristics similar to those found in pure hybrid duplexes. However, the DNA sugar puckers and the width and depth of the minor groove in the pure DNA section are more like B-form DNA, especially beyond the junction. Thus, this Okazaki fragment duplex assumes a conformation in solution that is a chimeric mixture of hybrid-form (H-form) and B-form structures and the overall molecule cannot be classified as either an A-form or a B-form duplex. The co-existence of these two different conformations in a single duplex gives rise to a structural discontinuity with a bend of approximately 18.1 (+/- 0.4) degrees at the junction between the hybrid and DNA segments that may be important for reverse transcriptase binding and RNase H cleavage of such molecules. Despite the fact that the solution structure is quite different from the all A-form structure reported recently for the exact same molecule in the crystalline state, a surprising number of local helical parameters were found to be quite similar to those reported for the crystal structure.
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PMID:The solution structure of the r(gcg)d(TATACCC):d(GGGTATACGC) Okazaki fragment contains two distinct duplex morphologies connected by a junction. 806 57

Ribonuclease H (Escherichia coli) contains one strong magnesium-binding site, as determined by metal-titration experiments monitored by high field 1H-NMR and also by direct titration calorimetry. Kinetic and thermodynamic parameters were evaluated by 25Mg-NMR and were as follows: dissociation constant Kd, approximately 60 +/- 10 microM; activation free energy delta G*, approximately 49.8 +/- 0.9 kJ; on/off-rate for magnesium binding Kon, approximately 1.8 x 10(8) M-1 s-1, koff, approximately 1.1 x 10(4) s-1; quadrupole coupling constant chi B, 1.2 +/- 0.2 MHz. The dissociation constant was independently determined by standard analysis of 1H chemical shifts in magnesium-titration experiments and by microcalorimetry (Kd approximately 200 +/- 20 microM). Cobalt hexaamine, which also activates RNase H [Jou, R. & Cowan, J. A. (1991) J. Am. Chem. Soc. 113, 6685-6686], appears to bind at the same location as Mg2+(aqueous). Assignments of C2H and C4H protons to specific histidine residues have been made by two-dimensional correlated spectroscopy experiments. Direct 25Mg-NMR pH titrations show that an ionizable residue (pKa approximately 5.8), most likely one of the carboxylates in the active site, influences magnesium binding. On the basis of the magnesium coordination chemistry elucidated herein, recent proposals on active-site chemistry are critically assessed and general physicochemical aspects of magnesium-binding sites on proteins and enzymes are discussed.
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PMID:Metallobiochemistry of the magnesium ion. Characterization of the essential metal-binding site in Escherichia coli ribonuclease H. 830 92

Assignments of 1H, 15N, and 13C magnetic resonances for ribonuclease H from Escherichia coli have been completed using double- and triple-resonance 2D and 3D NMR experiments. These assignments include all types of 1H, 15N, and 13C nuclei detectable by NMR. The enzyme used, which cleaves the RNA moiety of an RNA-DNA duplex, consists of 155 amino acid residues and has 1962 nuclei (227 nitrogen, 762 carbons, and 973 protons) observable independently by NMR. Among those, 1868 nuclei (95%) have been assigned. Two methods, 3D HCH and 13C-13C-1H heteroSQC/homoSQC, were newly devised to complete the side chain assignments. These methods were used to elucidate the -CH2- and -C-CH-substructures. Triple-resonance experiments to detect other types of substructures, (e.g., -N-CH- and -C-NH-) were also applied. In total, 10 kinds of 3D NMR experiments were used to complete the assignments. The chemical shifts obtained through the assignments were analyzed in terms of the tertiary structure of the protein molecule. Among the 13C chemical shifts, larger secondary shifts (deviations from shifts at the random coil state) were observed for the C alpha, C beta, and C' nuclei, which reflect the local structures on the backbone, that is, the alpha-helix, beta-sheet, and left-handed helix, respectively.
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PMID:Complete assignments of magnetic resonances of ribonuclease H from Escherichia coli by double- and triple-resonance 2D and 3D NMR spectroscopies. 838 89

Large quantities of RNA for study by NMR and X-ray crystallography can be produced by transcription reactions in vitro using T7 bacteriophage RNA polymerase. A limitation on producing RNA with this polymerase has been the strong dependence of the yield of the transcription reaction on the sequence at the 5' end of the RNA produced. We report a procedure for obtaining large quantities of enzymatically synthesized RNA from T7 RNA polymerase that has no dependence on the 5' end sequence of the target RNA. Ribonuclease H has been shown previously (Inoue H, Hayase Y, Iwai S, Ohtsuka E, 1987, FEBS Lett 215:327-330) to cleave RNA site specifically using 2'-O-methyl RNA/DNA chimeras to direct the cleavage site. We show that 2'-O-methyl RNA nucleotides on the 5'-side of the DNA nucleotides in the chimera are not essential for site-specific cleavage. This allowed us to design the method such that the same 2'-O-methyl chimera may be used to process any RNA sequence. We have adapted this reaction to the cleavage of NMR-scale quantities of RNA at high yield. RNA is synthesized using T7 RNA polymerase with a 15-nt high-yielding leader sequence at the 5' end, and then this sequence is cleaved off with the RNase H cleavage reaction. The cleaved RNA has 3'-hydroxyl and 5'-phosphate ends, so that the products can be used directly as substrates for ligation by T4 DNA ligase. We show that the cleavage reaction occurs efficiently in solution and on a solid streptavidin/agarose matrix. We report an example in which we are able to improve transcription yield by more than five-fold using this technique in the synthesis of a 15N isotopically labeled hairpin found in the Crithidia fasciculata spliced leader RNA. We are able to obtain a 0.5-mM NMR sample from this inherently poorly transcribing sequence, while minimizing the amount of isotopically labeled rNTPs used to produce it. The NMR spectroscopic results are consistent with the predicted RNA secondary structure.
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PMID:RNase H cleavage for processing of in vitro transcribed RNA for NMR studies and RNA ligation. 860 52

The protein fusion technique was applied in the synthesis of an artificial dimer of ribonuclease H (305 residues). 1H NMR spectroscopy was used to analyze the structure of this dimer. Spectral profiles and pKa values of the histidine residues obtained using 1H NMR indicate that the dimer retains the secondary and tertiary structures of the intact monomer. Selective spin-lattice relaxation measurements suggest that the two monomeric units in the dimer are in tight contact. Furthermore, the 2D 1H NMR and paramagnetic relaxation filter results show that the two monomers bind together through interactions between the N- and C-terminal sites of the linked regions.
J Biomol NMR 1996 Jan
PMID:Characterization of an artificial dimer of ribonuclease H using 1H NMR spectroscopy. 872 Aug 29

The three-dimensional solution structure of the hybrid-chimeric duplex r(gcca)d(CTGC).d(GCAGTGGC) has been determined by two-dimensional NMR, restrained molecular dynamics (rMD), and NOE back-calculation methods. This chimera, consisting of a chimeric RNA-DNA strand and its complementary DNA strand, is formed after priming (-)-strand DNA synthesis by tRNA(Lys3) and subsequent (+)-strand DNA synthesis by reverse transcriptase and is an obligatory intermediate in the formation of double-stranded DNA prior to HIV-1 retrovirus integration. The duplex consists of two different types of double helix: a hybrid form (H-form) and a B-form structure connected by a junction. It is chemically similar to several other Okazaki fragments whose structures have been previously determined in our laboratory. However, some structural parameters are not the same and were found to be sequence dependent. In particular, the sugar conformations at the DNA base pair proximal to the hybrid segment vary from O4'-endo to C2'-endo depending on the base composition. The position of the transition from the relatively wide groove of H-form to the narrow groove of B-form is also sequence dependent, occurring either exactly at the RNA-DNA junction or within the purely DNA segment of the chimera-as is the case in the structure of the present HIV-1 (-)-strand primer. This structural change produces a kink at the DNA-DNA step adjacent to the RNA-DNA junction in the HIV-1 (-)-strand primer. The sequence dependence of structures of RNA-DNA chimeric duplexes may be responsible for the variable cleavage pattern of different Okazaki fragments by reverse transcriptase RNase H.
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PMID:Structural variation among retroviral primer-DNA junctions: solution structure of the HIV-1 (-)-strand Okazaki fragment r(gcca)d(CTGC).d(GCAGTGGC). 878 May 9

The three-dimensional solution structure of the hybrid duplex r(gaggacug):d(CAGTCCTC) has been determined by two-dimensional NMR, distance geometry (DG), restrained molecular dynamics (rMD) and NOE back-calculation methods. This hybrid, consisting of a purine-rich RNA strand and a pyrimidine-rich DNA strand, is related to the polypurine (+)-strand primer formed after (-)-strand DNA synthesis and RNase H degradation of the viral RNA strand and contains the site of a specific cleavage by reverse transcription (RT) RNase H at the end of the HIV-1 polypurine tract. This polypurine primer is an important intermediate in the formation of virally encoded double-stranded DNA prior to HIV-1 retrovirus integration. The correct processing of this primer is vital in the life cycle of the human immunodeficiency virus type (HIV-1) retrovirus. The structure of the r(gaggacug):d(CAGTCCTC) hybrid, as determined in solution by NMR, is intermediate between canonical A-type and B-type double helices, and has mixed structural characteristics. It is quantitatively different from the previously determined solution structures of other RNA-DNA hybrids, particularly in the width and shape of the major groove, which is wider than the major groove of other hybrids and is close to the dimension of the major groove of B-type DNA duplexes. The structure of this hybrid duplex contains a prominent bend in the double helix with a magnitude and direction similar to the bend in Okazaki fragments. The structural features of the present duplex may explain the unique interactions of this sequence with HIV-1 RT during both (-)-strand and (+)-strand DNA synthesis.
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PMID:Solution structure of r(gaggacug):d(CAGTCCTC) hybrid: implications for the initiation of HIV-1 (+)-strand synthesis. 919 Oct 67

In this work, we present the first NMR solution structure of a DNA/RNA hybrid containing stereoregular Rp-phosphorothioate modifications of all DNA backbone linkages. The complex of the enzymatically synthesized phosphorothioate DNA octamer (all-Rp)-d(GCGTCAGG) and its complementary RNA r(CCUGACGC) was found to adopt an overall conformation within the A-form family. Most helical parameters and the sugar puckers of the DNA strand assume values intermediate between A- and B-form. The close structural similarity with the unmodified DNA/RNA hybrid of the same sequence may explain why both the natural and the sulfur-substituted complex can be recognized and digested by ribonuclease H.
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PMID:Structure of a stereoregular phosphorothioate DNA/RNA duplex. 954 16

A novel class of DNA-binding domains has been established from at least sixteen recently identified DNA-binding proteins. The three-dimensional structure of one of these domains, Mrf-2, has been solved using NMR methods. This structure is significantly different from known DNA-binding domain structures. The mechanism of DNA recognition by this motif has been suggested based on conserved residues, surface electrostatic potentials and chemical shift changes. This new DNA-binding motif shares structural homology with T4 RNase H, E. coli endonuclease III and Bacillus subtilis DNA polymerase I. The structural homology suggests a mechanism for substrate recognition by these enzymes.
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PMID:A novel DNA-binding motif shares structural homology to DNA replication and repair nucleases and polymerases. 980 40


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