Статті в журналах: "RNases H"

1

Allen, S. J. W., S. H. Krawczyk, L. R. McGee, N. Bischofberger, A. S. Mulato, and J. M. Cherrington. "Inhibition of HIV-1 RNase H Activity by Nucleotide Dimers and Monomers." Antiviral Chemistry and Chemotherapy 7, no. 1 (February 1996): 37–45. http://dx.doi.org/10.1177/095632029600700107.

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Nucleotide dimers and monomers were shown to inhibit human immunodeficiency virus type 1 (HIV) RNase H activity. Several effective inhibitors were identified and placed into three general groups based on biochemical characterization of their inhibition, The first group (group A) inhibited HIV RNase H and the closely related feline immunodeficiency virus (FIV) RNase H, but did not inhibit less related retroviral or cellular RNases H or HIV reverse transcriptase (RT). The second group (group B) inhibited the RNase H activity of several retroviruses as well as the reverse transcriptase function of HIV RT. The third group (group C) inhibited RNases H from retroviral and cellular sources but did not inhibit HIV RT. Kinetic analyses of HIV RNase H inhibition were conducted and all three types of inhibitors exhibited a competitive mode of inhibition with regard to substrate. The small nucleotides described here represent the most potent (Ki values from 0.57 to 16 μM) and selective inhibitors of HIV RNase H reported to date. Further structure - function analyses of these molecules may lead to the discovery of unique, potent antiretroviral therapeutics.

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2

Leich, Franziska, Nadine Stöhr, Anne Rietz, Renate Ulbrich-Hofmann, and Ulrich Arnold. "Endocytotic Internalization as a Crucial Factor for the Cytotoxicity of Ribonucleases." Journal of Biological Chemistry 282, no. 38 (July 17, 2007): 27640–46. http://dx.doi.org/10.1074/jbc.m702240200.

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The cytotoxic action of ribonucleases (RNases) requires the interaction of the enzyme with the cellular membrane, its internalization, translocation to the cytosol, and the degradation of ribonucleic acid. The interplay of these processes as well as the role of the thermodynamic and proteolytic stability, the catalytic activity, and the evasion from the intracellular ribonuclease inhibitor (RI) has not yet been fully elucidated. As cytosolic internalization is indispensable for the cytotoxicity of extracellular ribonucleases, we investigated the extent of cytosolic internalization of a cytotoxic, RI-evasive RNase A variant (G88R-RNase A) and of various similarly cytotoxic but RI-sensitive RNase A tandem enzyme variants in comparison to the internalization of the non-cytotoxic and RI-sensitive RNase A. After incubation of K-562 cells with the RNase A variants for 36 h, the internalized amount of RNases was analyzed by rapid cell disruption followed by subcellular fractionation and semiquantitative immunoblotting. The data indicate that an enhanced cellular uptake and an increased entry of the RNases into the cytosol can outweigh the abolishment of catalytic activity by RI. As all RNase A variants proved to be resistant to the proteases present in the different subcellular fractions for more than 100 h, our results suggest that the cytotoxic potency of RNases is determined by an efficient internalization into the cytosol.

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3

Watkins, Harriet A., and Edward N. Baker. "Structural and Functional Characterization of an RNase HI Domain from the Bifunctional Protein Rv2228c from Mycobacterium tuberculosis." Journal of Bacteriology 192, no. 11 (April 2, 2010): 2878–86. http://dx.doi.org/10.1128/jb.01615-09.

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ABSTRACT The open reading frame Rv2228c from Mycobacterium tuberculosis is predicted to encode a protein composed of two domains, each with individual functions, annotated through sequence similarity searches. The N-terminal domain is homologous with prokaryotic and eukaryotic RNase H domains and the C-terminal domain with α-ribazole phosphatase (CobC). The N-terminal domain of Rv2228c (Rv2228c/N) and the full-length protein were expressed as fusions with maltose binding protein (MBP). Rv2228c/N was shown to have RNase H activity with a hybrid RNA/DNA substrate as well as double-stranded RNase activity. The full-length protein was shown to have additional CobC activity. The crystal structure of the MBP-Rv2228c/N fusion protein was solved by molecular replacement and refined at 2.25-Å resolution (R = 0.182; R free = 0.238). The protein is monomeric in solution but associates in the crystal to form a dimer. The Rv2228c/N domain has the classic RNase H fold and catalytic machinery but lacks several surface features that play important roles in the cleavage of RNA/DNA hybrids by other RNases H. The absence of either the basic protrusion of some RNases H or the hybrid binding domain of others appears to be compensated by the C-terminal CobC domain in full-length Rv2228c. The double-stranded-RNase activity of Rv2228c/N contrasts with classical RNases H and is attributed to the absence in Rv2228c/N of a key phosphate binding pocket.

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4

Ohtani, Naoto, Mitsuru Haruki, Masaaki Morikawa, and Shigenori Kanaya. "Molecular diversities of RNases H." Journal of Bioscience and Bioengineering 88, no. 1 (January 1999): 12–19. http://dx.doi.org/10.1016/s1389-1723(99)80168-6.

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5

Hyjek, Malwina, Małgorzata Figiel, and Marcin Nowotny. "RNases H: Structure and mechanism." DNA Repair 84 (December 2019): 102672. http://dx.doi.org/10.1016/j.dnarep.2019.102672.

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6

Goulian, Mehran, and Cheryl J. Heard. "Discrimination between mammalian RNases H-1 and H-2." Analytical Biochemistry 192, no. 2 (February 1991): 398–402. http://dx.doi.org/10.1016/0003-2697(91)90555-8.

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7

Lim, Shion A., Kathryn M. Hart, Michael J. Harms, and Susan Marqusee. "Evolutionary trend toward kinetic stability in the folding trajectory of RNases H." Proceedings of the National Academy of Sciences 113, no. 46 (October 31, 2016): 13045–50. http://dx.doi.org/10.1073/pnas.1611781113.

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Proper folding of proteins is critical to producing the biological machinery essential for cellular function. The rates and energetics of a protein’s folding process, which is described by its energy landscape, are encoded in the amino acid sequence. Over the course of evolution, this landscape must be maintained such that the protein folds and remains folded over a biologically relevant time scale. How exactly a protein’s energy landscape is maintained or altered throughout evolution is unclear. To study how a protein’s energy landscape changed over time, we characterized the folding trajectories of ancestral proteins of the ribonuclease H (RNase H) family using ancestral sequence reconstruction to access the evolutionary history between RNases H from mesophilic and thermophilic bacteria. We found that despite large sequence divergence, the overall folding pathway is conserved over billions of years of evolution. There are robust trends in the rates of protein folding and unfolding; both modern RNases H evolved to be more kinetically stable than their most recent common ancestor. Finally, our study demonstrates how a partially folded intermediate provides a readily adaptable folding landscape by allowing the independent tuning of kinetics and thermodynamics.

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8

Hiller, Bjoern, Martin Achleitner, Silke Glage, Ronald Naumann, Rayk Behrendt, and Axel Roers. "Mammalian RNase H2 removes ribonucleotides from DNA to maintain genome integrity." Journal of Experimental Medicine 209, no. 8 (July 16, 2012): 1419–26. http://dx.doi.org/10.1084/jem.20120876.

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Ribonucleases H (RNases H) are endonucleases which cleave the RNA moiety of RNA/DNA hybrids. Their function in mammalian cells is incompletely understood. RNase H2 mutations cause Aicardi-Goutières syndrome, an inflammatory condition clinically overlapping with lupus erythematosus. We show that RNase H2 is essential in mouse embryonic development. RNase H2–deficient cells proliferated slower than control cells and accumulated in G2/M phase due to chronic activation of a DNA damage response associated with an increased frequency of single-strand breaks, increased histone H2AX phosphorylation, and induction of p53 target genes, most prominently the cyclin-dependent kinase inhibitor 1 encoding cell cycle inhibitor p21. RNase H2–deficient cells featured an increased genomic ribonucleotide load, suggesting that unrepaired ribonucleotides trigger the DNA damage response in these cells. Collectively, we show that RNase H2 is essential to remove ribonucleotides from the mammalian genome to prevent DNA damage.

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9

Kirby, Karen A., Bruno Marchand, Yee Tsuey Ong, Tanyaradzwa P. Ndongwe, Atsuko Hachiya, Eleftherios Michailidis, Maxwell D. Leslie, et al. "Structural and Inhibition Studies of the RNase H Function of Xenotropic Murine Leukemia Virus-Related Virus Reverse Transcriptase." Antimicrobial Agents and Chemotherapy 56, no. 4 (January 17, 2012): 2048–61. http://dx.doi.org/10.1128/aac.06000-11.

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ABSTRACTRNase H inhibitors (RNHIs) have gained attention as potential HIV-1 therapeutics. Although several RNHIs have been studied in the context of HIV-1 reverse transcriptase (RT) RNase H, there is no information on inhibitors that might affect the RNase H activity of other RTs. We performed biochemical, virological, crystallographic, and molecular modeling studies to compare the RNase H function and inhibition profiles of the gammaretroviral xenotropic murine leukemia virus-related virus (XMRV) and Moloney murine leukemia virus (MoMLV) RTs to those of HIV-1 RT. The RNase H activity of XMRV RT is significantly lower than that of HIV-1 RT and comparable to that of MoMLV RT. XMRV and MoMLV, but not HIV-1 RT, had optimal RNase H activities in the presence of Mn2+and not Mg2+. Using hydroxyl-radical footprinting assays, we demonstrated that the distance between the polymerase and RNase H domains in the MoMLV and XMRV RTs is longer than that in the HIV-1 RT by ∼3.4 Å. We identified one naphthyridinone and one hydroxyisoquinolinedione as potent inhibitors of HIV-1 and XMRV RT RNases H with 50% inhibitory concentrations ranging from ∼0.8 to 0.02 μM. Two acylhydrazones effective against HIV-1 RT RNase H were less potent against the XMRV enzyme. We also solved the crystal structure of an XMRV RNase H fragment at high resolution (1.5 Å) and determined the molecular details of the XMRV RNase H active site, thus providing a framework that would be useful for the design of antivirals that target RNase H.

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10

Cerritelli, Susana M., and Robert J. Crouch. "RNases H: Multiple roles in maintaining genome integrity." DNA Repair 84 (December 2019): 102742. http://dx.doi.org/10.1016/j.dnarep.2019.102742.

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11

OHTANI, Naoto, Hiroshi YANAGAWA, Masaru TOMITA, and Mitsuhiro ITAYA. "Identification of the first archaeal Type 1 RNase H gene from Halobacterium sp. NRC-1: archaeal RNase HI can cleave an RNA–DNA junction." Biochemical Journal 381, no. 3 (July 27, 2004): 795–802. http://dx.doi.org/10.1042/bj20040153.

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All the archaeal genomes sequenced to date contain a single Type 2 RNase H gene. We found that the genome of a halophilic archaeon, Halobacterium sp. NRC-1, contains an open reading frame with similarity to Type 1 RNase H. The protein encoded by the Vng0255c gene, possessed amino acid sequence identities of 33% with Escherichia coli RNase HI and 34% with a Bacillus subtilis RNase HI homologue. The B. subtilis RNase HI homologue, however, lacks amino acid sequences corresponding to a basic protrusion region of the E. coli RNase HI, and the Vng0255c has the similar deletion. As this deletion apparently conferred a complete loss of RNase H activity on the B. subtilis RNase HI homologue protein, the Vng0255c product was expected to exhibit no RNase H activity. However, the purified recombinant Vng0255c protein specifically cleaved an RNA strand of the RNA/DNA hybrid in vitro, and when the Vng0255c gene was expressed in an E. coli strain MIC2067 it could suppress the temperature-sensitive growth defect associated with the loss of RNase H enzymes of this strain. These results in vitro and in vivo strongly indicate that the Halobacterium Vng0255c is the first archaeal Type 1 RNase H. This enzyme, unlike other Type 1 RNases H, was able to cleave an Okazaki fragment-like substrate at the junction between the 3′-side of ribonucleotide and 5′-side of deoxyribonucleotide. It is likely that the archaeal Type 1 RNase H plays a role in the removal of the last ribonucleotide of the RNA primer from the Okazaki fragment during DNA replication.

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12

Stafford, Kate A., and Arthur G. Palmer III. "Evidence from molecular dynamics simulations of conformational preorganization in the ribonuclease H active site." F1000Research 3 (March 7, 2014): 67. http://dx.doi.org/10.12688/f1000research.3605.1.

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Ribonuclease H1 (RNase H) enzymes are well-conserved endonucleases that are present in all domains of life and are particularly important in the life cycle of retroviruses as domains within reverse transcriptase. Despite extensive study, especially of the E. coli homolog, the interaction of the highly negatively charged active site with catalytically required magnesium ions remains poorly understood. In this work, we describe molecular dynamics simulations of the E. coli homolog in complex with magnesium ions, as well as simulations of other homologs in their apo states. Collectively, these results suggest that the active site is highly rigid in the apo state of all homologs studied and is conformationally preorganized to favor the binding of a magnesium ion. Notably, representatives of bacterial, eukaryotic, and retroviral RNases H all exhibit similar active-site rigidity, suggesting that this dynamic feature is only subtly modulated by amino acid sequence and is primarily imposed by the distinctive RNase H protein fold.

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13

Schultz, Sharon J., Miaohua Zhang, and James J. Champoux. "Recognition of Internal Cleavage Sites by Retroviral RNases H." Journal of Molecular Biology 344, no. 3 (November 2004): 635–52. http://dx.doi.org/10.1016/j.jmb.2004.09.081.

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14

Ohtani, Naoto, Masaru Tomita, and Mitsuhiro Itaya. "Junction ribonuclease: a ribonuclease HII orthologue from Thermus thermophilus HB8 prefers the RNA–DNA junction to the RNA/DNA heteroduplex." Biochemical Journal 412, no. 3 (May 28, 2008): 517–26. http://dx.doi.org/10.1042/bj20080140.

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The genome of an extremely thermophilic bacterium, Thermus thermophilus HB8, contains a single ORF (open reading frame) encoding an RNase-HII-like sequence. Despite the presence of significant amino acid sequence identities with RNase (ribonuclease) HII enzymes, the ORF TTHA0198 could not suppress the temperature-sensitive growth defect of an RNase-H-deficient Escherichia coli mutant and the purified recombinant protein could not cleave an RNA strand of an RNA/DNA heteroduplex, suggesting that the TTHA0198 exhibited no RNase H activity both in vivo and in vitro. When oligomeric RNA–DNA/DNAs were used as a mimic substrate for Okazaki fragments, however, the protein cleaved them only at the 5′ side of the last ribonucleotide at the RNA–DNA junction. In fact, the TTHA0198 protein prefers the RNA–DNA junction to the RNA/DNA hybrid. We have referred to this activity as JRNase (junction RNase) activity, which recognizes an RNA–DNA junction of the RNA–DNA/DNA heteroduplex and cleaves it leaving a mono-ribonucleotide at the 5′ terminus of the RNA–DNA junction. E. coli and Deinococcus radiodurans RNases HII also cleaved the RNA–DNA/DNA substrates at the same site with a different metal-ion preference from that for RNase H activity, implying that the enzymes have JRNase activity as well as RNase H activity. The specialization in the JRNase activity of the RNase HII orthologue from T. thermophilus HB8 (Tth-JRNase) suggests that the JRNase activity of RNase HII enzymes might be independent of the RNase H activity.

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15

Lim, David, G. Glenn Gregorio, Craig Bingman, Erik Martinez-Hackert, Wayne A. Hendrickson, and Stephen P. Goff. "Crystal Structure of the Moloney Murine Leukemia Virus RNase H Domain." Journal of Virology 80, no. 17 (September 1, 2006): 8379–89. http://dx.doi.org/10.1128/jvi.00750-06.

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ABSTRACT A crystallographic study of the Moloney murine leukemia virus (Mo-MLV) RNase H domain was performed to provide information about its structure and mechanism of action. These efforts resulted in the crystallization of a mutant Mo-MLV RNase H lacking the putative helix C (ΔC). The 1.6-Å resolution structure resembles the known structures of the human immunodeficiency virus type 1 (HIV-1) and Escherichia coli RNase H. The structure revealed the coordination of a magnesium ion within the catalytic core comprised of the highly conserved acidic residues D524, E562, and D583. Surface charge mapping of the Mo-MLV structure revealed a high density of basic charges on one side of the enzyme. Using a model of the Mo-MLV structure superimposed upon a structure of HIV-1 reverse transcriptase bound to an RNA/DNA hybrid substrate, Mo-MLV RNase H secondary structures and individual amino acids were examined for their potential roles in binding substrate. Identified regions included Mo-MLV RNase H β1-β2, αA, and αB and residues from αB to αD and its following loop. Most of the identified substrate-binding residues corresponded with residues directly binding nucleotides in an RNase H from Bacillus halodurans as observed in a cocrystal structure with RNA/DNA. Finally, superimposition of RNases H of Mo-MLV, E. coli, and HIV-1 revealed that a loop of the HIV-1 connection domain resides within the same region of the Mo-MLV and E. coli C-helix. The HIV-1 connection domain may serve to recognize and bind the RNA/DNA substrate major groove.

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16

Zimmer, Anjali D., and Douglas Koshland. "Differential roles of the RNases H in preventing chromosome instability." Proceedings of the National Academy of Sciences 113, no. 43 (October 10, 2016): 12220–25. http://dx.doi.org/10.1073/pnas.1613448113.

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DNA:RNA hybrids can lead to DNA damage and genome instability. This damage can be prevented by degradation of the RNA in the hybrid by two evolutionarily conserved enzymes, RNase H1 and H2. Indeed, RNase H-deficient cells have increased chromosomal rearrangements. However, the quantitative and spatial contributions of the individual enzymes to hybrid removal have been unclear. Additionally, RNase H2 can remove single ribonucleotides misincorporated into DNA during replication. The relative contribution of DNA:RNA hybrids and misincorporated ribonucleotides to chromosome instability also was uncertain. To address these issues, we studied the frequency and location of loss-of-heterozygosity (LOH) events on chromosome III in Saccharomyces cerevisiae strains that were defective for RNase H1, H2, or both. We showed that RNase H2 plays the major role in preventing chromosome III instability through its hybrid-removal activity. Furthermore, RNase H2 acts pervasively at many hybrids along the chromosome. In contrast, RNase H1 acts to prevent LOH within a small region of chromosome III where the instability is dependent upon two hybrid-prone sequences. This restriction of RNase H1 activity to a subset of hybrids is not the result of its constrained localization, because we found it at hybrids genome-wide. This result suggests that the genome-protection activity of RNase H1 is regulated at a step after hybrid recognition. The global function of RNase H2 and the region-specific function of RNase H1 provide insight into why these enzymes with overlapping hybrid-removal activities have been conserved throughout evolution.

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17

Good-Avila, S. V., D. Majumder, H. Amos, and A. G. Stephenson. "Characterization of self-incompatibility in Campanula rapunculoides (Campanulaceae) through genetic analyses and microscopy." Botany 86, no. 1 (January 2008): 1–13. http://dx.doi.org/10.1139/b07-100.

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In this paper, we seek to identify the genetic basis of self-incompatibility (SI) in Campanula rapunculoides L. through diallel analysis of full siblings; to characterize the growth of pollen tubes in vivo after incompatible and compatible pollination; and to determine whether the SI system is based on pistil S-RNases. Pollinations were performed among individuals from five diallel crosses and scored for both fruit set and pollen-tube growth to determine the genetic basis of SI. On a subset of these individuals with known cross-(in)compatibility relationships, additional crosses were performed and pistils collected 1, 3, 6, 12, and 24 h after pollination to assess both the percentage of pollen grains that had germinated on the stigma, and the number of pollen tubes that had grown 20%, 40% 60%, 80%, and 100% of the distance down the pistil over five time intervals. Finally, total pistillate proteins were extracted and subjected to isoelectric focusing and RNase activity staining to find evidence of a highly basic S-RNases associated with SI in the Solanaceae. We found evidence that the SI system was based on the haplotype of the male gametophyte, and was not sporophytic. Protein analyses showed that SI was not based on a pistillate S-RNase. The existence of modifiers of SI and possible polyploidy at the S-locus complicated the expression of SI in this species, and single-gene inheritance could not be determined. This represents the first published characterization of incompatibility in the family Campanulaceae.

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18

Nowotny, Marcin, Sergei Gaidamakov, Robert J. Crouch, and Wei Yang. "Structural studies of RNases H and their complexes with RNA/DNA hybrids." Acta Crystallographica Section A Foundations of Crystallography 65, a1 (August 16, 2009): s138. http://dx.doi.org/10.1107/s0108767309097232.

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19

Ohtani, Naoto, Mitsuru Haruki, Masaaki Morikawa, Robert J. Crouch, Mitsuhiro Itaya, and Shigenori Kanaya. "Identification of the Genes Encoding Mn2+-Dependent RNase HII and Mg2+-Dependent RNase HIII fromBacillus subtilis: Classification of RNases H into Three Families†." Biochemistry 38, no. 2 (January 1999): 605–18. http://dx.doi.org/10.1021/bi982207z.

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20

An Lim, Shion, Kathryn M. Hart, Michael J. Harms, and Susan Marqusee. "An Evolutionary Trend towards Kinetic Stability in the Folding Trajectory of RNases H." Biophysical Journal 112, no. 3 (February 2017): 59a—60a. http://dx.doi.org/10.1016/j.bpj.2016.11.359.

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21

Permanasari, Etin-Diah, Kiyoshi Yasukawa, and Shigenori Kanaya. "Enzymatic Activities of RNase H Domains of HIV-1 Reverse Transcriptase with Substrate Binding Domains of Bacterial RNases H1 and H2." Molecular Biotechnology 57, no. 6 (February 12, 2015): 526–38. http://dx.doi.org/10.1007/s12033-015-9846-5.

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22

Hafkemeyer, Peter, Klaus Neftel, Reinhard Hobi, Andreas Pfaltz, Hans Lutz, Kersten Lüthi, Federico Focher, Silvio Spadari, and Ulrich Hübscher. "HP 0.35, a cephalosporin degradation product is a specific inhibitor of lentiviral RNAses H." Nucleic Acids Research 19, no. 15 (1991): 4059–65. http://dx.doi.org/10.1093/nar/19.15.4059.

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23

Rosen, Laura E., and Susan Marqusee. "Autonomously Folding Protein Fragments Reveal Differences in the Energy Landscapes of Homologous RNases H." PLOS ONE 10, no. 3 (March 24, 2015): e0119640. http://dx.doi.org/10.1371/journal.pone.0119640.

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24

Staroseletz, Yaroslav, Svetlana Gaponova, Olga Patutina, Elena Bichenkova, Bahareh Amirloo, Thomas Heyman, Daria Chiglintseva, and Marina Zenkova. "Site-Selective Artificial Ribonucleases: Renaissance of Oligonucleotide Conjugates for Irreversible Cleavage of RNA Sequences." Molecules 26, no. 6 (March 19, 2021): 1732. http://dx.doi.org/10.3390/molecules26061732.

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RNA-targeting therapeutics require highly efficient sequence-specific devices capable of RNA irreversible degradation in vivo. The most developed methods of sequence-specific RNA cleavage, such as siRNA or antisense oligonucleotides (ASO), are currently based on recruitment of either intracellular multi-protein complexes or enzymes, leaving alternative approaches (e.g., ribozymes and DNAzymes) far behind. Recently, site-selective artificial ribonucleases combining the oligonucleotide recognition motifs (or their structural analogues) and catalytically active groups in a single molecular scaffold have been proven to be a great competitor to siRNA and ASO. Using the most efficient catalytic groups, utilising both metal ion-dependent (Cu(II)-2,9-dimethylphenanthroline) and metal ion-free (Tris(2-aminobenzimidazole)) on the one hand and PNA as an RNA recognising oligonucleotide on the other, allowed site-selective artificial RNases to be created with half-lives of 0.5–1 h. Artificial RNases based on the catalytic peptide [(ArgLeu)2Gly]2 were able to take progress a step further by demonstrating an ability to cleave miRNA-21 in tumour cells and provide a significant reduction of tumour growth in mice.

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25

Smith, Robert M., Cherie M. Walton, Catherine H. Wu, and George Y. Wu. "Secondary Structure and Hybridization Accessibility of Hepatitis C Virus 3′-Terminal Sequences." Journal of Virology 76, no. 19 (October 1, 2002): 9563–74. http://dx.doi.org/10.1128/jvi.76.19.9563-9574.2002.

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ABSTRACT The 3′-terminal sequences of hepatitis C virus (HCV) positive- and negative-strand RNAs contribute cis-acting functions essential for viral replication. The secondary structure and protein-binding properties of these highly conserved regions are of interest not only for the further elucidation of HCV molecular biology, but also for the design of antisense therapeutic constructs. The RNA structure of the positive-strand 3′ untranslated region has been shown previously to influence binding by various host and viral proteins and is thus thought to promote HCV RNA synthesis and genome stability. Recent studies have attributed analogous functions to the negative-strand 3′ terminus. We evaluated the HCV negative-strand secondary structure by enzymatic probing with single-strand-specific RNases and thermodynamic modeling of RNA folding. The accessibility of both 3′-terminal sequences to hybridization by antisense constructs was evaluated by RNase H cleavage mapping in the presence of combinatorial oligodeoxynucleotide libraries. The mapping results facilitated identification of antisense oligodeoxynucleotides and a 10-23 deoxyribozyme active against the positive-strand 3′-X region RNA in vitro.

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26

Schultz, Sharon J., Miaohua Zhang, and James J. Champoux. "Sequence, Distance, and Accessibility Are Determinants of 5′-End-directed Cleavages by Retroviral RNases H." Journal of Biological Chemistry 281, no. 4 (November 22, 2005): 1943–55. http://dx.doi.org/10.1074/jbc.m510504200.

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27

Shen, Ying, Kyung Duk Koh, Bernard Weiss, and Francesca Storici. "Mispaired rNMPs in DNA are mutagenic and are targets of mismatch repair and RNases H." Nature Structural & Molecular Biology 19, no. 1 (December 4, 2011): 98–104. http://dx.doi.org/10.1038/nsmb.2176.

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28

Meng, Wenzhao, and Allen W. Nicholson. "Heterodimer-based analysis of subunit and domain contributions to double-stranded RNA processing by Escherichia coli RNase III in vitro." Biochemical Journal 410, no. 1 (January 29, 2008): 39–48. http://dx.doi.org/10.1042/bj20071047.

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Members of the RNase III family are the primary cellular agents of dsRNA (double-stranded RNA) processing. Bacterial RNases III function as homodimers and contain two dsRBDs (dsRNA-binding domains) and two catalytic sites. The potential for functional cross-talk between the catalytic sites and the requirement for both dsRBDs for processing activity are not known. It is shown that an Escherichia coli RNase III heterodimer that contains a single functional wt (wild-type) catalytic site and an inactive catalytic site (RNase III[E117A/wt]) cleaves a substrate with a single scissile bond with a kcat value that is one-half that of wt RNase III, but exhibits an unaltered Km. Moreover, RNase III[E117A/wt] cleavage of a substrate containing two scissile bonds generates singly cleaved intermediates that are only slowly cleaved at the remaining phosphodiester linkage, and in a manner that is sensitive to excess unlabelled substrate. These results demonstrate the equal probability, during a single binding event, of placement of a scissile bond in a functional or nonfunctional catalytic site of the heterodimer and reveal a requirement for substrate dissociation and rebinding for cleavage of both phosphodiester linkages by the mutant heterodimer. The rate of phosphodiester hydrolysis by RNase III[E117A/wt] has the same dependence on Mg2+ ion concentration as that of the wt enzyme, and exhibits a Hill coefficient (h) of 2.0±0.1, indicating that the metal ion dependence essentially reflects a single catalytic site that employs a two-Mg2+-ion mechanism. Whereas an E. coli RNase III mutant that lacks both dsRBDs is inactive, a heterodimer that contains a single dsRBD exhibits significant catalytic activity. These findings support a reaction pathway involving the largely independent action of the dsRBDs and the catalytic sites in substrate recognition and cleavage respectively.

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Li, Chang, Mengqi Lu, Junqin Zhou, Sen Wang, Yi Long, Yan Xu, and Xiaofeng Tan. "Transcriptome Analysis of the Late-Acting Self-Incompatibility Associated with RNase T2 Family in Camellia oleifera." Plants 12, no. 10 (May 9, 2023): 1932. http://dx.doi.org/10.3390/plants12101932.

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The Camellia oil tree (Camellia oleifera Abel.) is an important nonwood forest species in China, and the majority of its cultivars are late-acting self-incompatibility (LSI) types. Although several studies have examined the mechanism of LSI, the process is quite complicated and unclear. In this study, pollen tube growth and fruit setting of two Camellia oil tree cultivars Huashuo (HS) and Huajin (HJ) were investigated after non and self-pollination, and transcriptomic analysis of the ovaries was performed 48 h after self-pollination to identify the potential genes implicated in the LSI of Camellia oil trees. The results showed that the fruit set of HS was significantly higher than that of HJ after self-pollination. Transcriptomic analysis revealed that plant hormone signal transduction, the phosphatidylinositol signaling system, ATP-binding cassette (ABC) transporters, reactive oxygen species (ROS) metabolism, and Ca2+ signaling were mainly contributed in the LSI of reaction of Camellia oil tree. Moreover, nine RNase T2 genes were identified from the transcriptome analysis, which also showed that CoRNase7 participated in the self-incompatibility reaction in HS. Based on phylogenetic analysis, CoRNase6 was closely related to S-RNase from coffee, and CoRNase7 and CoRNase8 were closely related to S-RNase from Camellia sinensis. The 9 RNase T2 genes successfully produced proteins in prokaryotes. Subcellular localization indicated that CoRNase1 and CoRNase5 were cytoplasmic proteins, while CoRNase7 was a plasma membrane protein. These results screened the main metabolic pathways closely related to LSI in Camellia oil tree, and SI signal transduction might be regulated by a large molecular regulatory network. The discovery of T2 RNases provided evidence that Camellia oil tree might be under RNase-based gametophytic self-incompatibility.

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Schultz, Sharon J., Miaohua Zhang, and James J. Champoux. "Multiple Nucleotide Preferences Determine Cleavage-Site Recognition by the HIV-1 and M-MuLV RNases H." Journal of Molecular Biology 397, no. 1 (March 2010): 161–78. http://dx.doi.org/10.1016/j.jmb.2010.01.059.

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31

Schultz, Sharon J., Miaohua Zhang, and James J. Champoux. "Preferred Sequences within a Defined Cleavage Window Specify DNA 3′ End-directed Cleavages by Retroviral RNases H." Journal of Biological Chemistry 284, no. 47 (September 24, 2009): 32225–38. http://dx.doi.org/10.1074/jbc.m109.043158.

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32

Gugliotti, Lina A., Kiran B. Sakhuja, Hongsheng Wang, Julia Pinkhasov, Paul E. Love, Susana M. Cerritelli, Herbert Morse, and Robert J. Crouch. "Constitutive Lymphoid Expression of the Nuclear Form of RNase H1 Is Associated with a Developmental Bottleneck at the Pro-B Cell Stage of B Cell Differentiation." Blood 114, no. 22 (November 20, 2009): 4702. http://dx.doi.org/10.1182/blood.v114.22.4702.4702.

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Abstract Abstract 4702 The development of B lymphocytes and the process of lineage determination are initiated by expression of a set of transcriptional regulators leading to V(D)J recombination events initiated by double-strand DNA breaks. Subsequently, these recombinations form DNAs that permit transcription of immunoglobulin genes and translation of the corresponding mRNAs, first by joining the V(D)J DNA sequences, then by recombination, that generates various isotypes of immunoglobulins by class-switch recombination (CSR). Formation of R-loops, regions containing RNA/DNA hybrid and a displaced single-stranded DNA, have been shown to lead to recombination in bacteria, yeast, HeLa and chick cells. Expression in each of these cases of excess ribonuclease H1 (RNase H1), a class of enzymes that degrade RNA in RNA/DNA hybrids, has ameliorated the deleterious effects and decreased recombinational events associated with R-loop formation. R-loops have been observed following transcription of the switch regions that occurs during CSR. The possibility that R-loops are important in V(D)J recombination has not been addressed, and whether ribonucleases H (RNases H) play a role in this process is still uncertain. Transgenic (TG) mice that overexpress RNase H1 in B and T cells (M27F7) were employed in this study. FACS analysis of hematopoietic cells from TG mice revealed a decrease in pre-B cells in the bone marrow. The data indicate a block at the pro-B to pre-B stage of B cell development, which may be the result of apoptosis due to the failure to generate a productive VH-D-JH rearrangement and expression of the pre-B cell receptor. A few B cells that successfully passed these checkpoints predominately differentiated into marginal zone and B1a cells in the peripheral lymphoid organs of the TG mice. These data suggest that R-loops are important in H chain gene rearrangement. This research is supported by the Intramural Research Program of the National Institutes of Health, the Eunice Kennedy Shriver National Institute of Child Health and Human Development and the National Institute of Allergy and Infectious Diseases. Disclosures: No relevant conflicts of interest to declare.

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Guo, Yan, Jie Wu, Shilin Zhao, Fei Ye, Yinghao Su, Travis Clark, Quanhu Sheng, Brian Lehmann, Xiao-ou Shu, and Qiuyin Cai. "RNA Sequencing of Formalin-Fixed, Paraffin-Embedded Specimens for Gene Expression Quantification and Data Mining." International Journal of Genomics 2016 (2016): 1–10. http://dx.doi.org/10.1155/2016/9837310.

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Background. Proper rRNA depletion is crucial for the successful utilization of FFPE specimens when studying gene expression. We performed a study to evaluate two major rRNA depletion methods: Ribo-Zero and RNase H. RNAs extracted from 4 samples were treated with the two rRNA depletion methods in duplicate and sequenced (N=16). We evaluated their reducibility, ability to detect RNA, and ability to molecularly subtype these triple negative breast cancer specimens.Results. Both rRNA depletion methods produced consistent data between the technical replicates. We found that the RNase H method produced higher quality RNAseq data as compared to the Ribo-Zero method. In addition, we evaluated the RNAseq data generated from the FFPE tissue samples for noncoding RNA, including lncRNA, enhancer/super enhancer RNA, and single nucleotide variation (SNV). We found that the RNase H is more suitable for detecting high-quality, noncoding RNAs as compared to the Ribo-Zero and provided more consistent molecular subtype identification between replicates. Unfortunately, neither method produced reliable SNV data.Conclusions. In conclusion, for FFPE specimens, the RNase H rRNA depletion method performed better than the Ribo-Zero. Neither method generates data sufficient for SNV detection.

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Morris, Shannon, and Jonathan Leis. "Changes in Rous Sarcoma Virus RNA Secondary Structure near the Primer Binding Site upon tRNATrpPrimer Annealing." Journal of Virology 73, no. 8 (August 1, 1999): 6307–18. http://dx.doi.org/10.1128/jvi.73.8.6307-6318.1999.

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ABSTRACT Predicted secondary-structure elements encompassing the primer binding site in the 5′ untranslated region of Rous sarcoma virus (RSV) RNA play an integral role in multiple viral replications steps including reverse transcription, DNA integration, and RNA packaging (A. Aiyar, D. Cobrinik, Z. Ge, H. J. Kung, and J. Leis, J. Virol. 66:2464–2472, 1992; D. Cobrinik, A. Aiyar, Z. Ge, M. Katzman, H. Huang, and J. Leis, J. Virol. 65:3864–3872, 1991; J. T. Miller, Z. Ge, S. Morris, K. Das, and J. Leis, J. Virol. 71:7648–7656, 1997). These elements include the U5-Leader stem, U5-IR stem-loop, and U5-TΨC interaction region. Limited digestion of the 5′ untranslated region of wild-type and mutant RSV RNAs with structure- and/or sequence-specific RNases detects the presence of the U5-Leader stem and the U5-IR stem-loop. When a tRNATrp primer is annealed to wild-type RNAs in vitro, limited nuclease mapping indicates that the U5-IR stem becomes partially unwound. This is not observed when mutant RNAs with altered U5-IR stem-loop structures are substituted for wild-type RNAs. The U5-Leader stem also becomes destabilized when the tRNA primer is annealed to either wild-type or mutant RNA fragments. Nuclease mapping studies of tRNATrp, as well as the viral RNA, indicate that the U5-TΨC helix does form in vitro upon primer annealing. Collectively, these data suggest that the various structural elements near the RSV primer binding site undergo significant changes during the process of primer annealing.

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35

Dharap, Ashuthosh, Kellie Bowen, Robert Place, Long-Cheng Li, and Raghu Vemuganti. "Transient Focal Ischemia Induces Extensive Temporal Changes in Rat Cerebral MicroRNAome." Journal of Cerebral Blood Flow & Metabolism 29, no. 4 (January 14, 2009): 675–87. http://dx.doi.org/10.1038/jcbfm.2008.157.

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MicroRNAs (miRNAs) are ∼22 nucleotides long, noncoding RNAs that control cellular function by either degrading mRNAs or arresting their translation. To understand their functional significance in ischemic pathophysiology, we profiled miRNAs in adult rat brain as a function of reperfusion time after transient middle cerebral artery occlusion. Of the 238 miRNAs evaluated, 8 showed increased and 12 showed decreased expression at least at 4 out of 5 reperfusion time points studied between 3 h and 3 days compared with sham. Of those, 17 showed > 5 fold change. Bioinformatics analysis indicated a correlation between miRNAs altered to several mRNAs known to mediate inflammation, transcription, neuroprotection, receptors function, and ionic homeostasis. Antagomir-mediated prevention of mir-145 expression led to an increased protein expression of its downstream target superoxide dismutase-2 in the postischemic brain. In silico analysis showed sequence complementarity of eight miRNAs induced after focal ischemia to 877 promoters indicating the possibility of noncoding RNA-induced activation of gene expression. The mRNA expression of the RNases Drosha and Dicer, cofactor Pasha, and the pre-miRNA transporter exportin-5, which modulate miRNA biogenesis, were not altered after transient middle cerebral artery occlusion. Thus, the present studies indicate a critical role of miRNAs in controlling mRNA transcription and translation in the postischemic brain.

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36

Gruber, Cornelia, Torsten Gursinsky, Selma Gago-Zachert, Vitantonio Pantaleo, and Sven-Erik Behrens. "Effective Antiviral Application of Antisense in Plants by Exploiting Accessible Sites in the Target RNA." International Journal of Molecular Sciences 24, no. 24 (December 5, 2023): 17153. http://dx.doi.org/10.3390/ijms242417153.

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Antisense oligodeoxynucleotides (ASOs) have long been used to selectively inhibit or modulate gene expression at the RNA level, and some ASOs are approved for clinical use. However, the practicability of antisense technologies remains limited by the difficulty of reliably predicting the sites accessible to ASOs in complex folded RNAs. Recently, we applied a plant-based method that reproduces RNA-induced RNA silencing in vitro to reliably identify sites in target RNAs that are accessible to small interfering RNA (siRNA)-guided Argonaute endonucleases. Here, we show that this method is also suitable for identifying ASOs that are effective in DNA-induced RNA silencing by RNases H. We show that ASOs identified in this way that target a viral genome are comparably effective in protecting plants from infection as siRNAs with the corresponding sequence. The antiviral activity of the ASOs could be further enhanced by chemical modification. This led to two important conclusions: siRNAs and ASOs that can effectively knock down complex RNA molecules can be identified using the same approach, and ASOs optimized in this way could find application in crop protection. The technology developed here could be useful not only for effective RNA silencing in plants but also in other organisms.

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Lu, Gaofeng, Elena Lomonosova, Xiaohong Cheng, Eileen A. Moran, Marvin J. Meyers, Stuart F. J. Le Grice, Craig J. Thomas, et al. "Hydroxylated Tropolones Inhibit Hepatitis B Virus Replication by Blocking Viral Ribonuclease H Activity." Antimicrobial Agents and Chemotherapy 59, no. 2 (December 1, 2014): 1070–79. http://dx.doi.org/10.1128/aac.04617-14.

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ABSTRACTHepatitis B virus (HBV) remains a major human pathogen despite the development of both antiviral drugs and a vaccine, in part because the current therapies do not suppress HBV replication far enough to eradicate the virus. Here, we screened 51 troponoid compounds for their ability to suppress HBV RNaseH activity and HBV replication based on the activities of α-hydroxytropolones against HIV RNaseH, with the goal of determining whether the tropolone pharmacophore may be a promising scaffold for anti-HBV drug development. Thirteen compounds inhibited HBV RNaseH, with the best 50% inhibitory concentration (IC50) being 2.3 μM. Similar inhibition patterns were observed against HBV genotype D and C RNaseHs, implying limited genotype specificity. Six of 10 compounds tested against HBV replication in culture suppressed replication via blocking of viral RNaseH activity, with the best 50% effective concentration (EC50) being 0.34 μM. Eighteen compounds inhibited recombinant human RNaseH1, and moderate cytotoxicity was observed for all compounds (50% cytotoxic concentration [CC50] = 25 to 79 μM). Therapeutic indexes ranged from 3.8 to 94. Efficient inhibition required an intact α-hydroxytropolone moiety plus one or more short appendages on the tropolone ring, but a wide variety of constituents were permissible. These data indicate that troponoids and specifically α-hydroxytropolones are promising lead candidates for development as anti-HBV drugs, providing that toxicity can be minimized. Potential anti-RNaseH drugs are envisioned to be employed in combination with the existing nucleos(t)ide analogs to suppress HBV replication far enough to block genomic maintenance, with the goal of eradicating infection.

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Arudchandran, Arulvathani, Susana Cerritelli, Scott Narimatsu, Mitsuhiro Itaya, Deug-Yong Shin, Yuji Shimada, and Robert Crouch. "The absence of ribonuclease H1 or H2 alters the sensitivity of Saccharomyces cerevisiae to hydroxyurea, caffeine and ethyl methanesulphonate: implications for roles of RNases H in DNA replication and repair." Genes to Cells 5, no. 10 (October 2000): 789–802. http://dx.doi.org/10.1046/j.1365-2443.2000.00373.x.

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39

Sharma, Vasudha, Prachi Thakore, and Sharmistha Majumdar. "THAP9 Transposase Cleaves DNA via Conserved Acidic Residues in an RNaseH-Like Domain." Cells 10, no. 6 (May 29, 2021): 1351. http://dx.doi.org/10.3390/cells10061351.

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The catalytic domain of most ‘cut and paste’ DNA transposases have the canonical RNase-H fold, which is also shared by other polynucleotidyl transferases such as the retroviral integrases and the RAG1 subunit of V(D)J recombinase. The RNase-H fold is a mixture of beta sheets and alpha helices with three acidic residues (Asp, Asp, Glu/Asp—DDE/D) that are involved in the metal-mediated cleavage and subsequent integration of DNA. Human THAP9 (hTHAP9), homologous to the well-studied Drosophila P-element transposase (DmTNP), is an active DNA transposase that, although domesticated, still retains the catalytic activity to mobilize transposons. In this study we have modeled the structure of hTHAP9 using the recently available cryo-EM structure of DmTNP as a template to identify an RNase-H like fold along with important acidic residues in its catalytic domain. Site-directed mutagenesis of the predicted catalytic residues followed by screening for DNA excision and integration activity has led to the identification of candidate Ds and Es in the RNaseH fold that may be a part of the catalytic triad in hTHAP9. This study has helped widen our knowledge about the catalytic activity of a functionally uncharacterized transposon-derived gene in the human genome.

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Corona, Angela, Anna Schneider, Kristian Schweimer, Paul Rösch, Birgitta M. Wöhrl, and Enzo Tramontano. "Inhibition of Foamy Virus Reverse Transcriptase by Human Immunodeficiency Virus Type 1 RNase H Inhibitors." Antimicrobial Agents and Chemotherapy 58, no. 7 (May 5, 2014): 4086–93. http://dx.doi.org/10.1128/aac.00056-14.

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ABSTRACTRNase H plays an essential role in the replication of human immunodeficiency virus type 1 (HIV-1). Therefore, it is a promising target for drug development. However, the identification of HIV-1 RNase H inhibitors (RHIs) has been hampered by the open morphology of its active site, the limited number of available RNase H crystal structures in complex with inhibitors, and the fact that, due to the high concentrations of Mg2+needed for protein stability, HIV-1 RNase H is not suitable for nuclear magnetic resonance (NMR) inhibitor studies. We recently showed that the RNase H domains of HIV-1 and prototype foamy virus (PFV) reverse transcriptases (RTs) exhibit a high degree of structural similarity. Thus, we examined whether PFV RNase H can serve as an HIV-1 RNase H model for inhibitor interaction studies. Five HIV-1 RHIs inhibited PFV RNase H activity at low-micromolar concentrations similar to those of HIV-1 RNase H, suggesting pocket similarity of the RNase H domains. NMR titration experiments with the PFV RNase H domain and the RHI RDS1643 (6-[1-(4-fluorophenyl)methyl-1H-pyrrol-2-yl)]-2,4-dioxo-5-hexenoic acid ethyl ester) were performed to determine its binding site. Based on these results and previous data,in silicodocking analysis showed a putative RDS1643 binding region that reaches into the PFV RNase H active site. Structural overlays were performed with HIV-1 and PFV RNase H to propose the RDS1643 binding site in HIV-1 RNase H. Our results suggest that this approach can be used to establish PFV RNase H as a model system for HIV-1 RNase H in order to identify putative inhibitor binding sites in HIV-1 RNase H.

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Corona, Angela, Francesco Saverio Di Leva, Sylvain Thierry, Luca Pescatori, Giuliana Cuzzucoli Crucitti, Frederic Subra, Olivier Delelis, et al. "Identification of Highly Conserved Residues Involved in Inhibition of HIV-1 RNase H Function by Diketo Acid Derivatives." Antimicrobial Agents and Chemotherapy 58, no. 10 (August 4, 2014): 6101–10. http://dx.doi.org/10.1128/aac.03605-14.

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ABSTRACTHIV-1 reverse transcriptase (RT)-associated RNase H activity is an essential function in viral genome retrotranscription. RNase H is a promising drug target for which no inhibitor is available for therapy. Diketo acid (DKA) derivatives are active site Mg2+-binding inhibitors of both HIV-1 RNase H and integrase (IN) activities. To investigate the DKA binding site of RNase H and the mechanism of action, six couples of ester and acid DKAs, derived from 6-[1-(4-fluorophenyl)methyl-1H-pyrrol-2-yl)]-2,4-dioxo-5-hexenoic acid ethyl ester (RDS1643), were synthesized and tested on both RNase H and IN functions. Most of the ester derivatives showed selectivity for HIV-1 RNase H versus IN, while acids inhibited both functions. Molecular modeling and site-directed mutagenesis studies on the RNase H domain demonstrated different binding poses for ester and acid DKAs and proved that DKAs interact with residues (R448, N474, Q475, Y501, and R557) involved not in the catalytic motif but in highly conserved portions of the RNase H primer grip motif. The ester derivative RDS1759 selectively inhibited RNase H activity and viral replication in the low micromolar range, making contacts with residues Q475, N474, and Y501. Quantitative PCR studies and fluorescence-activated cell sorting (FACS) analyses showed that RDS1759 selectively inhibited reverse transcription in cell-based assays. Overall, we provide the first demonstration that RNase H inhibition by DKAs is due not only to their chelating properties but also to specific interactions with highly conserved amino acid residues in the RNase H domain, leading to effective targeting of HIV retrotranscription in cells and hence offering important insights for the rational design of RNase H inhibitors.

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Wang, Yafang, Namin Hu, Chang Liu, Cunpeng Nie, Manman He, Juan Zhang, Qiaoqin Yu, Chuan Zhao, Tingting Chen, and Xia Chu. "An RNase H-powered DNA walking machine for sensitive detection of RNase H and the screening of related inhibitors." Nanoscale 12, no. 3 (2020): 1673–79. http://dx.doi.org/10.1039/c9nr07550j.

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43

Delviks-Frankenberry, Krista A., Galina N. Nikolenko, Rebekah Barr, and Vinay K. Pathak. "Mutations in Human Immunodeficiency Virus Type 1 RNase H Primer Grip Enhance 3′-Azido-3′-Deoxythymidine Resistance." Journal of Virology 81, no. 13 (April 11, 2007): 6837–45. http://dx.doi.org/10.1128/jvi.02820-06.

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ABSTRACT We recently observed that mutations in the human immunodeficiency type 1 (HIV-1) reverse transcriptase (RT) connection domain significantly increase 3′-azido-3′-deoxythymidine (AZT) resistance up to 536 times over wild-type (WT) RT in the presence of thymidine analog resistance mutations (TAMs). These mutations also decreased RT template switching, suggesting that they altered the balance between nucleotide excision and template RNA degradation, which in turn increased AZT resistance. Several residues in the HIV-1 connection domain contact the primer strand and form an RNase H primer grip structure that helps to position the primer-template at the RNase H and polymerase active sites. To test the hypothesis that connection domain mutations enhanced AZT resistance by influencing the RNase H primer grip, we determined the effects of alanine substitutions in RNase H primer grip residues on nucleoside RT inhibitor resistance in the context of a WT, TAM-containing, or K65R-containing polymerase domain. Ten of the 11 RNase H primer grip mutations increased AZT resistance 20 to 243 times above WT levels in the context of a TAM-containing polymerase domain. Furthermore, all mutations in the RNase H primer grip decreased template switching, suggesting that they reduced RNase H activity. These results demonstrate that mutations in the RNase H primer grip region can significantly enhance AZT resistance and support the hypothesis that mutations in the connection and RNase H domains can increase resistance by altering the RNase H primer grip region, changing interactions between RT and the template-primer complex and/or shifting the balance between the polymerase and RNase H activities.

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Lee, Hyunjee, HyeokJin Cho, Jooyoung Kim, Sua Lee, Jungmin Yoo, Daeho Park, and Gwangrog Lee. "RNase H is an exo- and endoribonuclease with asymmetric directionality, depending on the binding mode to the structural variants of RNA:DNA hybrids." Nucleic Acids Research 50, no. 4 (November 12, 2021): 1801–14. http://dx.doi.org/10.1093/nar/gkab1064.

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Abstract RNase H is involved in fundamental cellular processes and is responsible for removing the short stretch of RNA from Okazaki fragments and the long stretch of RNA from R-loops. Defects in RNase H lead to embryo lethality in mice and Aicardi-Goutieres syndrome in humans, suggesting the importance of RNase H. To date, RNase H is known to be a non-sequence-specific endonuclease, but it is not known whether it performs other functions on the structural variants of RNA:DNA hybrids. Here, we used Escherichia coli RNase H as a model, and examined its catalytic mechanism and its substrate recognition modes, using single-molecule FRET. We discovered that RNase H acts as a processive exoribonuclease on the 3′ DNA overhang side but as a distributive non-sequence-specific endonuclease on the 5′ DNA overhang side of RNA:DNA hybrids or on blunt-ended hybrids. The high affinity of previously unidentified double-stranded (ds) and single-stranded (ss) DNA junctions flanking RNA:DNA hybrids may help RNase H find the hybrid substrates in long genomic DNA. Our study provides new insights into the multifunctionality of RNase H, elucidating unprecedented roles of junctions and ssDNA overhang on RNA:DNA hybrids.

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45

Farias, Richard V., Deborah A. Vargas, Andres E. Castillo, Beatriz Valenzuela, Marie L. Coté, Monica J. Roth, and Oscar Leon. "Expression of an Mg2+-Dependent HIV-1 RNase H Construct for Drug Screening." Antimicrobial Agents and Chemotherapy 55, no. 10 (July 18, 2011): 4735–41. http://dx.doi.org/10.1128/aac.00658-11.

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ABSTRACTA single polypeptide of the HIV-1 reverse transcriptase that reconstituted Mg2+-dependent RNase H activity has been made. Using molecular modeling, the construct was designed to encode the p51 subunit joined by a linker to the thumb (T), connection (C), and RNase H (R) domains of p66. This p51-G-TCR construct was purified from the soluble fraction of anEscherichia colistrain, MIC2067(DE3), lacking endogenous RNase HI and HII. The p51-G-TCR RNase H construct displayed Mg2+-dependent activity using a fluorescent nonspecific assay and showed the same cleavage pattern as HIV-1 reverse transcriptase (RT) on substrates that mimic the tRNA removal required for second-strand transfer reactions. The mutant E706Q (E478Q in RT) was purified under similar conditions and was not active. The RNase H of the p51-G-TCR RNase H construct and wild type HIV-1 RT had similarKms for an RNA-DNA hybrid substrate and showed similar inhibition kinetics to two known inhibitors of the HIV-1 RT RNase H.

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46

Brincat, Jennifer L., Julie K. Pfeiffer, and Alice Telesnitsky. "RNase H Activity Is Required for High-Frequency Repeat Deletion during Moloney Murine Leukemia Virus Replication." Journal of Virology 76, no. 1 (January 1, 2002): 88–95. http://dx.doi.org/10.1128/jvi.76.1.88-95.2002.

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ABSTRACT It has been postulated that retroviral recombination, like strong stop template switching, requires the RNase H activity of reverse transcriptase. To address this hypothesis, Moloney murine leukemia virus-based vectors, which were designed to test the recombination-related property of direct repeat deletion, were encapsidated in virions engineered to contain phenotypic mixtures of wild-type and RNase H catalytic site point mutant reverse transcriptase. Integrated provirus titers per milliliter were determined for these phenotypically mixed virions, and vector proviruses were screened to determine what percentage contained repeat deletions. The results revealed a steady decline in direct repeat deletion frequency that correlated with decreases in functional RNase H, with greater than fourfold decreases in repeat deletion frequency observed when 95% of virion reverse transcriptase was RNase H defective. Parallel experiments were performed to address effects of molar excesses of RNase H relative to functional DNA polymerase. These experiments demonstrated that increasing the stoichiometry of RNase H relative to the amount of functional DNA polymerase had minimal effects on direct repeat deletion frequency. DNA synthesis was error prone when directed principally by RNase H mutant reverse transcriptase, suggesting a role for RNase H catalytic integrity in the fidelity of intracellular reverse transcription.

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47

Keck, James L., Eric R. Goedken, and Susan Marqusee. "Activation/Attenuation Model for RNase H." Journal of Biological Chemistry 273, no. 51 (December 18, 1998): 34128–33. http://dx.doi.org/10.1074/jbc.273.51.34128.

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48

Krakowiak, Agnieszka, Alina Owczarek, Maria Koziołkiewicz, and Wojciech J. Stec. "Stereochemical Course ofEscherichia coli RNase H." ChemBioChem 3, no. 12 (December 2, 2002): 1242–50. http://dx.doi.org/10.1002/1439-7633(20021202)3:12<1242::aid-cbic1242>3.0.co;2-y.

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49

Su, Hua-Poo, Youwei Yan, G. Sridhar Prasad, Robert F. Smith, Christopher L. Daniels, Pravien D. Abeywickrema, John C. Reid, et al. "Structural Basis for the Inhibition of RNase H Activity of HIV-1 Reverse Transcriptase by RNase H Active Site-Directed Inhibitors." Journal of Virology 84, no. 15 (May 19, 2010): 7625–33. http://dx.doi.org/10.1128/jvi.00353-10.

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ABSTRACT HIV/AIDS continues to be a menace to public health. Several drugs currently on the market have successfully improved the ability to manage the viral burden in infected patients. However, new drugs are needed to combat the rapid emergence of mutated forms of the virus that are resistant to existing therapies. Currently, approved drugs target three of the four major enzyme activities encoded by the virus that are critical to the HIV life cycle. Although a number of inhibitors of HIV RNase H activity have been reported, few inhibit by directly engaging the RNase H active site. Here, we describe structures of naphthyridinone-containing inhibitors bound to the RNase H active site. This class of compounds binds to the active site via two metal ions that are coordinated by catalytic site residues, D443, E478, D498, and D549. The directionality of the naphthyridinone pharmacophore is restricted by the ordering of D549 and H539 in the RNase H domain. In addition, one of the naphthyridinone-based compounds was found to bind at a second site close to the polymerase active site and non-nucleoside/nucleotide inhibitor sites in a metal-independent manner. Further characterization, using fluorescence-based thermal denaturation and a crystal structure of the isolated RNase H domain reveals that this compound can also bind the RNase H site and retains the metal-dependent binding mode of this class of molecules. These structures provide a means for structurally guided design of novel RNase H inhibitors.

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Corona, Angela, Valentina Onnis, Claudia Del Vecchio, Francesca Esposito, Yung-Chi Cheng, and Enzo Tramontano. "2-(Arylamino)-6-(trifluoromethyl)nicotinic Acid Derivatives: New HIV-1 RT Dual Inhibitors Active on Viral Replication." Molecules 25, no. 6 (March 15, 2020): 1338. http://dx.doi.org/10.3390/molecules25061338.

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The persistence of the AIDS epidemic, and the life-long treatment required, indicate the constant need of novel HIV-1 inhibitors. In this scenario the HIV-1 Reverse Transcriptase (RT)-associated ribonuclease H (RNase H) function is a promising drug target. Here we report a series of compounds, developed on the 2-amino-6-(trifluoromethyl)nicotinic acid scaffold, studied as promising RNase H dual inhibitors. Among the 44 tested compounds, 34 inhibited HIV-1 RT-associated RNase H function in the low micromolar range, and seven of them showed also to inhibit viral replication in cell-based assays with a selectivity index up to 10. The most promising compound, 21, inhibited RNase H function with an IC50 of 14 µM and HIV-1 replication in cell-based assays with a selectivity index greater than 10. Mode of action studies revealed that compound 21 is an allosteric dual-site compound inhibiting both HIV-1 RT functions, blocking the polymerase function also in presence of mutations carried by circulating variants resistant to non-nucleoside inhibitors, and the RNase H function interacting with conserved regions within the RNase H domain. Proving compound 21 as a promising lead for the design of new allosteric RNase H inhibitors active against viral replication with not significant cytotoxic effects.

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