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)

Central the regulation and mechanism of class switch recombination is the understanding of the relationship between transcription and DNA recombination. We demonstrated previously, using mini-chromosome substrates, that physiologically oriented transcription is required for recombination to occur between switch regions. In this report, we demonstrate the formation of an RNA:DNA complex under in vitro transcription conditions for these same and other switch DNA fragments. We find that cell-free transcription of repetitive murine switch regions (Smu, S gamma 2b and S gamma 3) leads to altered DNA mobility on agarose gels. These altered mobilities are resistant to RNase A but sensitive to RNase H. Transcription in the presence of labeled ribonucleotides demonstrates the stable physical association of the RNA with the DNA. Importantly, complex formation only occurs upon transcription in the physiologic orientation. Reaban and Griffin [1990 Nature, 348, 342-344] found an RNA:DNA hybrid structure that was limited to an atypical 143 nucleotide purine region within a 2.3 kb S alpha segment. Here we demonstrate RNA:DNA hybrid formation in more typical switch sequences (lacking the atypical 143 nucleotide purine tract) from a variety of switch regions that are only 60-70% purine on the non-template strand. These results suggest a general model involving an RNA:DNA complex as an intermediate during class switch recombination.
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PMID:RNA:DNA complex formation upon transcription of immunoglobulin switch regions: implications for the mechanism and regulation of class switch recombination. 855 58

We have recently found that stable R-loop formation occurs in vivo and in vitro when a portion of the Escherichia coli rrnB operon is transcribed preferentially in its physiological orientation. Our results also suggested that the formation of such structures was more frequent in topA mutants and was sensitive to the template DNA supercoiling level. In the present report we investigated in greater detail the involvement of DNA topoisomerases in this process. By using an in vitro transcription system with phage RNA polymerases, we found that hypernegative supercoiling of plasmid DNAs in the presence of DNA gyrase is totally abolished by RNase H, suggesting that extensive R-looping occurs during transcription in the presence of DNA gyrase. When RNase A is present, significant hypernegative supercoiling occurs only when the 567-base pair rrnB HindIII fragment is transcribed in its physiological orientation. This result suggests that more stable R-loops are being produced in this orientation. Our results also suggest that DNA gyrase can participate in the process of R-loop elongation. The strong transcription-induced relaxing activity of E. coli DNA topoisomerase I is shown to efficiently counteract the effect of DNA gyrase and thus inhibit extensive R-looping. In addition, we found that an R-looped plasmid DNA is a better substrate for relaxation by E. coli DNA topoisomerase I as compared with a non-R-looped substrate.
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PMID:Roles of DNA topoisomerases in the regulation of R-loop formation in vitro. 899 16

The Ig heavy chain class switch in B lymphocytes involves a unique genetic recombination that fuses specific regions within the Ig locus and deletes intervening sequences. Here we describe a novel exonuclease activity in nuclear lysates of B cells in an in vitro assay. This activity was induced in B lymphocytes after treatment with either LPSs or CD40 ligand/anti-delta-dextran, both of which induce switch recombination, and considerably less activity was detected in untreated or anti-delta-dextran-treated B cells, Con A-stimulated spleen cells, liver cells, or a number of cell lines. The exonuclease activity was dependent on divalent cations, and both 3' and 5' labels were efficiently removed from DNA substrates. The presence of RNase A, but not RNase H, inhibited exonucleolytic digestion, suggesting that a ribonucleoprotein is responsible for the exonucleolysis. The DNA digestion appears to be nonspecific, since DNA substrates with either switch-mu or unrelated sequence were hydrolyzed with comparable efficiency. Germ-line switch region transcripts (Ig gamma1, Ig gamma3, and Ig alpha) strongly inhibited the exonucleolysis of switch-mu DNA but not that of unrelated control DNA, while switch antisense RNA or tRNA were much less effective inhibitors.
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PMID:Stimulation of murine B lymphocytes induces a DNA exonuclease whose activity on switch-mu DNA is specifically inhibited by other germ-line switch region RNAs. 953 Dec 92

Availability of 4-thiouridine (4-thioU)-containing RNAs is the prerequisite for 4-thioU site-specific cross-linking studies. This paper presents a method for constructing such RNAs. A 5'- and a 3'-RNA are synthesized via phage RNA polymerase transcription and/or RNase H site-specific cleavage directed by 2'-O-methyl-RNA-DNA chimeras. These two half-RNAs in combination correspond to the sequence of full-length RNA, with a single nucleotide gap at the junction that will be filled in with a 4-thiouridylate. A single p4SUp, which is derived from 4SUpN (N can be any nucleotide) via 5'-phosphorylation (therefore, the phosphate can be radioactive) followed by RNase A digestion, is then ligated to the 3' end of the 5'-half RNA with T4 RNA ligase. The 3'-phosphate of the ligated product is subsequently removed by calf intestinal alkaline phosphatase to produce a 3'-hydroxyl group. The resulting 5'-half RNA and the 3'-half RNA with a 5'-phosphate group (which can also be radioactive) are then aligned with a bridging deoxyoligonucleotide and ligated with T4 DNA ligase. This method was previously applied to the P120 pre-mRNA that contains an AT-AC intron, yielding three RNAs each containing a single 4-thioU near the 5'-splice site. Subsequent cross-linking studies with these RNAs yielded detailed information regarding interactions between the 5'-splice site and other spliceosomal snRNAs and between the 5'-splice site and proteins during splicing. Because there is no sequence constraint surrounding the site of 4-thioU substitution, this method should be applicable to many other RNAs.
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PMID:Construction of 4-thiouridine site-specifically substituted RNAs for cross-linking studies. 1020 12

Spectroscopic and calorimetric techniques were employed to characterize and contrast the binding of the aminoglycoside paromomycin to three octamer nucleic acid duplexes of identical sequence but different strand composition (a DNA.RNA hybrid duplex and the corresponding DNA.DNA and RNA.RNA duplexes). In addition, the impact of paromomycin binding on both RNase H- and RNase A-mediated cleavage of the RNA strand in the DNA.RNA duplex was also determined. Our results reveal the following significant features: (i) Paromomycin binding enhances the thermal stabilities of the RNA.RNA and DNA.RNA duplexes to similar extents, with this thermal enhancement being substantially greater in magnitude than that of the DNA.DNA duplex. (ii) Paromomycin binding to the DNA.RNA hybrid duplex induces CD changes consistent with a shift from an A-like to a more canonical A-conformation. (iii) Paromomycin binding to all three octamer duplexes is linked to the uptake of a similar number of protons, with the magnitude of this number being dependent on pH. (iv) The affinity of paromomycin for the three host duplexes follows the hierarchy, RNA.RNA > DNA.RNA >> DNA.DNA. (v) The observed affinity of paromomycin for the RNA.RNA and DNA.RNA duplexes decreases with increasing pH. (vi) The binding of paromomycin to the DNA.RNA hybrid duplex inhibits both RNase H- and RNase A-mediated cleavage of the RNA strand. We discuss the implications of our combined results with regard to the specific targeting of DNA.RNA hybrid duplex domains and potential antiretroviral applications.
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PMID:Aminoglycoside complexation with a DNA.RNA hybrid duplex: the thermodynamics of recognition and inhibition of RNA processing enzymes. 1278 87

Human ribonuclease-1 (hRNase-1) is an extracellular enzyme found in exocrine pancreas, blood, milk, saliva, urine and seminal plasma, which has been implicated in digestion of dietary RNA and in antiviral host defense. The enzyme is characterized by a high catalytic activity toward both single-stranded and double-stranded RNA. In this study, we explored the possibility that hRNase-1 may also be provided with a ribonuclease H activity, i.e. be able to digest the RNA component of RNA:DNA hybrids. For this purpose, we developed an accurate and sensitive real-time RNase H assay based on a fluorogenic substrate made of a 12 nt 5'-fluorescein-labeled RNA hybridized to a complementary 3'-quencher-modified DNA. Under physiological-like conditions, hRNase-1 was found to cleave the RNA:DNA hybrid very efficiently, as expressed by a kcat/K(m) of 330 000 M(-1) s(-1), a value that is over 180-fold higher than that obtained with the homologous bovine RNase A and only 8-fold lower than that measured with Escherichia coli RNase H. The kinetic characterization of hRNase-1 showed that its hybridase activity is maximal at neutral pH, increases with lowering ionic strength and is fully inhibited by the cytosolic RNase inhibitor. Overall, the reported data widen our knowledge of the enzymatic properties of hRNase-1 and provide new elements for the comprehension of its biological function.
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PMID:Hybridase activity of human ribonuclease-1 revealed by a real-time fluorometric assay. 1673 29

DNA-RNase H adducts were used for site specific cleavage of RNA and DNA-RNA duplexes, whereas nonspecific DNA interaction with ribonuclease A (RNase A) has been observed. The aim of this study was to examine the complexation of calf-thymus DNA with RNase A at physiological condition, using constant DNA concentration (12.5 mM) and various protein contents (1 microM to 270 microM). FTIR, UV-visible, and CD spectroscopic methods were used to analyse protein binding mode, the binding constant and the effects of nucleic acid-enzyme interaction on both DNA and protein conformations. Our structural analysis showed a strong RNase-PO2 binding and minor interaction with G-C bases with overall binding constant of K = 6.1 x 10(4) M(-1). The RNase-DNA interaction alters the protein secondary structure with a major reduction of the alpha-helix and increase of the beta-sheet and random structure, while DNA remains in the B-family structure.
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PMID:DNA interaction with RNase A alters protein conformation. 1726 94

The circular bacterial genome DNA exists in cells in the form of nucleoids. In the present study, using genetic, molecular and structural biology techniques, we show that nascent single-stranded RNAs are involved in the step-wise folding of nucleoid fibers. In Escherichia coli, RNase A degraded thicker fibers (30 and 80 nm wide) into thinner fibers (10 nm wide), while RNase III and RNase H degraded 80-nm fibers into 30-nm (but not 10-nm) fibers. Similarly in Staphylococcus aureus, RNase A treatment resulted in 10-nm fibers. Treatment with the transcription inhibitor, rifampicin, in the absence of RNase A changed most nucleoid fibers to 10-nm fibers. Proteinase-K treatment of nucleoids exposed DNA. Thus, the smallest structural unit is an RNase A-resistant 10-nm fiber composed of DNA and proteins, and the hierarchical structure of the bacterial chromosome is controlled by transcription itself. In addition, the formation of 80-nm fibers from 30-nm fibers requires double-stranded RNA and RNA-DNA hetero duplex. RNA is evident in the architecture of log-phase uncondensed and stationary-phase condensed nucleoids.
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PMID:Transcription-coupled nucleoid architecture in bacteria. 1790 74

Ribonucleases (RNases) with different sequence specificities are used for a variety of analytical purposes, including RNA sequencing, mapping, and quantitation. One very common application for RNase A is presented in this unit and involves hydrolyzing RNA that contaminates DNA preparations. Two other commonly used RNases, RNase H and RNase T1, are also described. In addition, many commercially available RNases are sequence-specific endoribonucleases, a property has been used for enzymatic sequencing of RNA.
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PMID:Ribonucleases. 1826 22

Ribonuclease enzymes (RNases) play key roles in the maturation and metabolism of all RNA molecules. Computational simulations of the processes involved can help to elucidate the underlying enzymatic mechanism and is often employed in a synergistic approach together with biochemical experiments. Theoretical calculations require atomistic details regarding the starting geometries of the molecules involved, which, in the absence of crystallographic data, can only be achieved from computational docking studies. Fortunately, docking algorithms have improved tremendously in recent years, so that reliable structures of enzyme-ligand complexes can now be successfully obtained from computation. However, most docking programs are not particularly optimized for nucleotide docking. In order to assist our studies on the cleavage of RNA by the two most important ribonuclease enzymes, RNase A and RNase H, we evaluated four docking tools-MOE2009, Glide 5.5, QXP-Flo+0802, and Autodock 4.0-for their ability to simulate complexes between these enzymes and RNA oligomers. To validate our results, we analyzed the docking results with respect to the known key interactions between the protein and the nucleotide. In addition, we compared the predicted complexes with X-ray structures of the mutated enzyme as well as with structures obtained from previous calculations. In this manner, we were able to prepare the desired reaction state complex so that it could be used as the starting structure for further DFT/B3LYP QM/MM reaction mechanism studies.
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PMID:Nucleotide docking: prediction of reactant state complexes for ribonuclease enzymes. 2112 May 56


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