Biomedical subjects

Robert J Crouch

Publications and source records attributed to Robert J Crouch.

10 recordsLinked to original sources

PubMed2005

Crystal structures of RNase H bound to an RNA/DNA hybrid: substrate specificity and metal-dependent catalysis.

RNase H belongs to a nucleotidyl-transferase superfamily, which includes transposase, retroviral integrase, Holliday junction resolvase, and RISC nuclease Argonaute. We report the crystal structures of RNase H complexed with an RNA/DNA hybrid and a mechanism for substrate recognition and two-metal-ion-dependent catalysis. RNase H specifically recognizes the A form RNA strand and the B form DNA strand. Structure comparisons lead us to predict the catalytic residues of Argonaute and conclude that two-metal-ion catalysis is a general feature of the superfamily. In nucleases, the two metal ions are asymmetrically coordinated and have distinct roles in activating the nucleophile and stabilizing the transition state. In transposases, they are symmetrically coordinated and exchange roles to alternately activate a water and a 3'-OH for successive strand cleavage and transfer by a ping-pong mechanism.

Arabidopsis Proteins↗

PubMed2005

Eukaryotic RNases H1 act processively by interactions through the duplex RNA-binding domain.

Ribonucleases H have mostly been implicated in eliminating short RNA primers used for initiation of lagging strand DNA synthesis. Escherichia coli RNase HI cleaves these RNA-DNA hybrids in a distributive manner. We report here that eukaryotic RNases H1 have evolved to be processive enzymes by attaching a duplex RNA-binding domain to the RNase H region. Highly conserved amino acids of the duplex RNA-binding domain are required for processivity and nucleic acid binding, which leads to dimerization of the protein. The need for a processive enzyme underscores the importance in eukaryotic cells of processing long hybrids, most of which remain to be identified. However, long RNA-DNA hybrids formed during immunoglobulin class-switch recombination are potential targets for RNase H1 in the nucleus. In mitochondria, where RNase H1 is essential for DNA formation during embryogenesis, long hybrids may be involved in DNA replication.

Amino Acid Sequence↗

PubMed2005

Selective inhibition of HIV-1 reverse transcriptase-associated ribonuclease H activity by hydroxylated tropolones.

High-throughput screening of a National Cancer Institute library of pure natural products identified the hydroxylated tropolone derivatives beta-thujaplicinol (2,7-dihydroxy-4-1(methylethyl)-2,4,6-cycloheptatrien-1-one) and manicol (1,2,3,4-tetrahydro-5-7-dihydroxy-9-methyl-2-(1-methylethenyl)-6H-benzocyclohepten-6-one) as potent and selective inhibitors of the ribonuclease H (RNase H) activity of human immunodeficiency virus-type 1 reverse transcriptase (HIV-1 RT). beta-Thujaplicinol inhibited HIV-1 RNase H in vitro with an IC50 of 0.2 microM, while the IC50 for Escherichia coli and human RNases H was 50 microM and 5.7 microM, respectively. In contrast, the related tropolone analog beta-thujaplicin (2-hydroxy-4-(methylethyl)-2,4,6-cycloheptatrien-1-one), which lacks the 7-OH group of the heptatriene ring, was inactive, while manicol, which possesses a 7-OH group, inhibited HIV-1 and E.coli RNases H with IC50 = 1.5 microM and 40 microM, respectively. Such a result highlights the importance of the 2,7-dihydroxy function of these tropolone analogs, possibly through a role in metal chelation at the RNase H active site. Inhibition of HIV-2 RT-associated RNase H indirectly indicates that these compounds do not occupy the nonnucleoside inhibitor-binding pocket in the vicinity of the DNA polymerase domain. Both beta-thujaplicinol and manicol failed to inhibit DNA-dependent DNA polymerase activity of HIV-1 RT at a concentration of 50 microM, suggesting that they are specific for the C-terminal RNase H domain, while surface plasmon resonance studies indicated that the inhibition was not due to intercalation of the analog into the nucleic acid substrate. Finally, we have demonstrated synergy between beta-thujaplicinol and calanolide A, a nonnucleoside inhibitor of HIV-1 RT, raising the possibility that both enzymatic activities of HIV-1 RT can be simultaneously targeted.

Anti-HIV Agents↗

PubMed2004

A capillary electrophoretic assay for ribonuclease H activity.

A capillary electrophoretic assay was developed to measure the ribonuclease (RNase) H activity of human immunodeficiency virus (HIV) type 1 reverse transcriptase. Cleavage of a fluorescein-labeled RNA-DNA heteroduplex was monitored by capillary electrophoresis. This new assay was used as a secondary assay to confirm hits from a high-throughput screening program. Since autofluorescent compounds in samples migrated differently from both substrate and product in most cases, the assay was extremely robust for assaying enzymatic inhibition of such samples, in contrast to a simple well-based approach. The assay was broadly applicable to other RNases H, specifically those from human, Escherichia coli, and HIV-2, although product profiles varied for each enzyme.

Base Sequence↗

PubMed2004

RNase H2 of Saccharomyces cerevisiae is a complex of three proteins.

The composition of RNase H2 has been a long-standing problem. Whereas bacterial and archaeal RNases H2 are active as single polypeptides, the Saccharomyces cerevisiae homolog, Rnh2Ap, when expressed in Escherichia coli, fails to produce an active RNase H2. By affinity chromatography purification and identification of polypeptides associated with a tagged S.cerevisiae Rnh2Ap, we obtained a complex of three proteins [Rnh2Ap (Rnh201p), Ydr279p (Rnh202p) and Ylr154p (Rnh203p)] that together are necessary and sufficient for RNase H2 activity [correction]. Deletion of the gene encoding any one of the proteins or mutations in the catalytic site in Rnh2A led to loss of RNase H2 activity. Even when S.cerevisiae RNase H2 is catalytically compromised, it still exhibits a preference for cleavage of the phosphodiester bond on the 5' side of a ribonucleotide-deoxyribonucleotide sequence in substrates mimicking RNA-primed Okazaki fragments or a single ribonucleotide embedded in a duplex DNA. Interestingly, Ydr279p and Ylr154p have homologous proteins only in closely related species. The multisubunit nature of S.cerevisiae RNase H2 may be important both for structural purposes and to provide a means of interacting with other proteins involved in DNA replication/repair and transcription.

Binding Sites↗

PubMed2004

Effects of RNA polymerase modifications on transcription-induced negative supercoiling and associated R-loop formation.

Transcription in the absence of topoisomerase I, but in the presence of DNA gyrase, can result in the formation of hypernegatively supercoiled DNA and associated R-loops. In this paper, we have used several strategies to study the effects of elongation/termination properties of RNA polymerase on such transcription-induced supercoiling. Effects on R-loop formation were exacerbated when cells were exposed to translation inhibitors, a condition that stimulated the accumulation of R-loop-dependent hypernegative supercoiling. Translation inhibitors were not acting by decreasing (p)ppGpp levels as the absence of (p)ppGpp in spoT relA mutant strains had little effect on hypernegative supercoiling. However, an rpoB mutation leading to the accumulation of truncated RNAs considerably reduced R-loop-dependent hypernegative supercoiling. Transcription of an rrnB fragment preceded by a mutated and inactive boxA sequence to abolish the rrnB antitermination system also considerably reduced R-loop-dependent supercoiling. Taken together, our results indicate that RNA polymerase elongation/termination properties can have a major impact on R-loop-dependent supercoiling. We discuss different possibilities by which RNA polymerase directly or indirectly participates in R-loop formation in Escherichia coli. Finally, our results also indicate that what determines the steady-state level of hypernegatively supercoiled DNA in topA null mutants is likely to be complex and involves a multitude of factors, including the status of RNA polymerase, transcription-translation coupling, the cellular level of RNase HI, the status of DNA gyrase and the rate of relaxation of supercoiled DNA.

Bacterial Proteins↗

PubMed2003

Selective inhibitory DNA aptamers of the human RNase H1.

Human RNase H1 binds double-stranded RNA via its N-terminal domain and RNA-DNA hybrid via its C-terminal RNase H domain, the latter being closely related to Escherichia coli RNase HI. Using SELEX, we have generated a set of DNA sequences that can bind efficiently (K(d) values ranging from 10 to 80 nM) to the human RNase H1. None of them could fold into a simple perfect double-stranded DNA hairpin confirming that double-stranded DNA does not constitute a trivial ligand for the enzyme. Only two of the 37 DNA aptamers selected were inhibitors of human RNase H1 activity. The two inhibitory oligomers, V-2 and VI-2, were quite different in structure with V-2 folding into a large, imperfect but stable hairpin loop. The VI-2 structure consists of a central region unimolecular quadruplex formed by stacking of two guanine quartets flanked by the 5' and 3' tails that form a stem of six base pairs. Base pairing between the 5' and 3' tails appears crucial for conferring the inhibitory properties to the aptamer. Finally, the inhibitory aptamers were capable of completely abolishing the action of an antisense oligonucleotide in a rabbit reticulocyte lysate supplemented with human RNase H1, with IC50 ranging from 50 to 100 nM.

Animals↗

PubMed2003

Humanized docking system for assembly of targeting drug delivery complexes.

Targeted drug delivery requires 'loading' drugs onto targeting proteins. Traditional technologies for loading drugs rely on chemical conjugation of drugs or drug carriers to targeting proteins. An alternative approach might rely on assembly of targeting complexes using a docking system that includes two components: a 'docking' tag fused to a targeting protein, and a 'payload' module containing an adapter protein for non-covalent binding to the docking tag. We describe here a fully humanized adapter/docking tag system based on non-covalent interaction between two fragments of human pancreatic RNase I. A 15 amino acid long N-terminal fragment of RNase I designed to serve as a docking tag, was fused to the N-terminus of human vascular endothelial growth factor that served as a targeting protein. An 18-125 and an 18-127 amino acid long fragments of RNase I were engineered, expressed and refolded into active conformations to serve as adapter proteins. Interactions between the targeting and adapter proteins were characterized using enzymatic analysis and surface plasmon resonance. Targeting DNA delivery complexes were assembled, characterized by dynamic light scattering, and found to be very effective in receptor-mediated DNA delivery.

Cell Line↗

PubMed2003

Failure to produce mitochondrial DNA results in embryonic lethality in Rnaseh1 null mice.

Although ribonucleases H (RNases H) have long been implicated in DNA metabolism, they are not required for viability in prokaryotes or unicellular eukaryotes. We generated Rnaseh1(-/-) mice to investigate the role of RNase H1 in mammals and observed developmental arrest at E8.5 in null embryos. A fraction of the mainly nuclear RNase H1 was targeted to mitochondria, and its absence in embryos resulted in a significant decrease in mitochondrial DNA content, leading to apoptotic cell death. This report links RNase H1 to generation of mitochondrial DNA, providing direct support for the strand-coupled mechanism of mitochondrial DNA replication. These findings also have important implications for therapy of mitochondrial dysfunctions and drug development for the structurally related RNase H of HIV.

Alleles↗

PubMed2002

Multiple ribonuclease H-encoding genes in the Caenorhabditis elegans genome contrasts with the two typical ribonuclease H-encoding genes in the human genome.

Database searches of the Caenorhabditis elegans and human genomic DNA sequences revealed genes encoding ribonuclease H1 (RNase H1) and RNase H2 in each genome. The human genome contains a single copy of each gene, whereas C. elegans has four genes encoding RNase H1-related proteins and one gene for RNase H2. By analyzing the mRNAs produced from the C. elegans genes, examining the amino acid sequence of the predicted protein, and expressing the proteins in Esherichia coli we have identified two active RNase H1-like proteins. One is similar to other eukaryotic RNases H1, whereas the second RNase H (rnh-1.1) is unique. The rnh-1.0 gene is transcribed as a dicistronic message with three dsRNA-binding domains; the mature mRNA is transspliced with SL2 splice leader and contains only one dsRNA-binding domain. Formation of RNase H1 is further regulated by differential cis-splicing events. A single rnh-2 gene, encoding a protein similar to several other eukaryotic RNase H2L's, also has been examined. The diversity and enzymatic properties of RNase H homologues are other examples of expansion of protein families in C. elegans. The presence of two RNases H1 in C. elegans suggests that two enzymes are required in this rather simple organism to perform the functions that are accomplished by a single enzyme in more complex organisms. Phylogenetic analysis indicates that the active C. elegans RNases H1 are distantly related to one another and that the C. elegans RNase H1 is more closely related to the human RNase H1. The database searches also suggest that RNase H domains of LTR-retrotransposons in C. elegans are quite unrelated to cellular RNases H1, but numerous RNase H domains of human endogenous retroviruses are more closely related to cellular RNases H.

Amino Acid Sequence↗