Disease relevance of RNASEH1

• Unlike the E. coli enzyme, human RNase H1 displays a strong positional preference for cleavage, i.e. it cleaves between 8 and 12 nucleotides from the 5'-RNA-3'-DNA terminus of the duplex [1].
• The intracellular localization of the enzymes, assayed by green-fluorescent protein fusions, showed that RNase H1 was present throughout the whole cell for all cell types analyzed, whereas RNase H2 was restricted to the nucleus in all cells except the prostate cancer line 15PC3 that expressed the protein throughout the cell [2].
• These findings also have important implications for therapy of mitochondrial dysfunctions and drug development for the structurally related RNase H of HIV [3].
• Reverse transcriptase with concomitant ribonuclease H activity in the cell-free synthesis of branched RNA-linked msDNA of Myxococcus xanthus [4].
• HIV-1 RT-associated ribonuclease H displays both endonuclease and 3'----5' exonuclease activity [5].

High impact information on RNASEH1

• Stable overexpression of RNase H suppressed the DNA-fragmentation and hypermutation phenotypes [6].
• Upon removal of the first eight nucleotides of the U1 snRNA in the particles by site-directed hydrolysis with ribonuclease H in the presence of a synthetic complementary oligodeoxynucleotide splicing is completely abolished [7].
• Depletion of U22 from Xenopus oocytes by oligonucleotide-directed ribonuclease H targeting prevented the processing of 18S ribosomal RNA (rRNA) at both ends [8].
• This report links RNase H1 to generation of mitochondrial DNA, providing direct support for the strand-coupled mechanism of mitochondrial DNA replication [3].
• 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 [3].

Chemical compound and disease context of RNASEH1

• Here we show that SBC ODNs strand invade a hairpin in the mini-exon RNA of Leishmania amazonensis and that the resulting heteroduplexes are substrates for Escherichia coli RNase H. SBC ODNs either with phosphodiester or phosphorothioate backbones form more stable hybrids with RNA than normal base (NB) ODNs [9].
• The RNase H activity of a mutant HIV-1 RT with serine at position 280 is completely resistant to inactivation by the three thiol reactive compounds we tested, which demonstrates that cysteine 280 is the critical residue [10].
• Biochemical studies on the reverse transcriptase and RNase H activities from human immunodeficiency virus strains resistant to 3'-azido-3'-deoxythymidine [11].
• Substitution of serine or alanine at position 445 (which lies in the RNase H domain) diminishes, but does not abolish, the RNase H activity of HIV-2 without affecting polymerase activity [10].
• Localization of the active site of HIV-1 reverse transcriptase-associated RNase H domain on a DNA template using site-specific generated hydroxyl radicals [12].

Biological context of RNASEH1

• Structural requirements at the catalytic site of the heteroduplex substrate for human RNase H1 catalysis [13].
• The human RNase H1 cleavage patterns observed for heteroduplexes with various 3'-DNA/5'-RNA and 5'-DNA/3'-RNA termini indicate that the 5'-most cleavage site on the oligoribonucleotide is positioned 7 bp from the first 3'-DNA/5'-RNA base pair [14].
• Ribonuclease H1 maps to chromosome 2 and has at least three pseudogene loci in the human genome [15].
• The cDNAs for human RNASEH1 and mouse Rnaseh1 were obtained, their nucleotide sequences determined, and the proteins expressed in Escherichia coli and partially purified [16].
• Although they share a similar overall fold related to ribonucleases of the RNase H family, they have local differences at the putative active site [17].

Anatomical context of RNASEH1

• Whole cell extracts of the cell lines yielded similar RNase H cleavage activity in an in vitro liquid assay, in contrast to the efficacy of the ODNs in vivo [2].
• We have demonstrated that the 60S complex represents the assembly of two single splicing complexes on the individual introns by conversion of the 60S double splicing complexes into single 50S spliceosomes by oligodeoxynucleotide directed RNase H cleavage of the double-intron pre-mRNAs within the middle exon [18].
• Oligodeoxynucleotides containing phosphodiester or modified internucleoside linkages were investigated with respect to their ability to be acted on by ribonuclease H activities present in a HeLa cell nuclear extract after hybridization with complementary sequences in RNA [19].
• In the present study, the effects of DNA uracilation on (-) strand DNA synthesis, RNase H activity, and (+) strand DNA synthesis were investigated in a cell-free system [20].
• Cellular RNase H from calf thymus and RNase H-II from Rauscher leukemia virus are likewise resistant to omicron-phenanthroline inhibition, implying non-involvement of zinc in the nucleic acid hydrolysis by these enzymes [21].

Associations of RNASEH1 with chemical compounds

• The oxidized and NEM alkylated forms of human RNase H1 exhibited binding affinities for the heteroduplex substrate comparable with the reduced form of the enzyme [22].
• The cysteine residues responsible for the observed redox-dependent activity of human RNase H1 were determined by site-directed mutagenesis to involve Cys(147) and Cys(148) [22].
• Comparison of the kcat, Km, and Kd for the alanine-substituted mutants with those for human RNase H1 suggests that Lys59 and Lys60 are involved in binding to the heteroduplex and that Trp43 is responsible for properly positioning the enzyme on the substrate for catalysis [14].
• Human RNase H1 uses one tryptophan and two lysines to position the enzyme at the 3'-DNA/5'-RNA terminus of the heteroduplex substrate [14].
• The presence or absence of phosphate or hydroxyl groups at either the 3'-DNA or 5'-RNA terminus had no effect on the human RNase H1 cleavage pattern [14].

Physical interactions of RNASEH1

• In one, occurring during minus strand synthesis, positioning of the RNase H is determined by binding of the polymerase active site to the DNA 3'-end [23].

Enzymatic interactions of RNASEH1

• Following second-strand synthesis by means of RNase H-induced nick translation by DNA polymerase I the overall yields in double-stranded cDNA were slightly higher when unfractionated cytoplasmic RNA was used as starting template [24].
• Additionally, the RNA component of the oligo/ RNA duplex is efficiently cleaved by RNase H, the site of endonucleolytic cleavage being dictated by the length of the oligodeoxynucleoside phosphorothioate segment [25].

Regulatory relationships of RNASEH1

• They were not able to compete with the polymerase and inhibited reverse transcription only through RNase H-mediated cleavage of the target [26].
• These observations not only indicate that PKC-alpha protein expression can be down-regulated by both RNase H-dependent and -independent mechanisms but also that down-regulation of PKC-alpha is insufficient by itself to "chemosensitize" cells [27].

Other interactions of RNASEH1

• Further studies by RNase H digestion revealed the presence of smaller TRH transcripts in the hamster testes than those in the rat testis [28].
• Following the identification of accessible ribozyme target sites by RNase H mapping, several hammerhead ribozymes were generated that cleave with comparable efficiency two different splice forms of cyclin E mRNA and the full-length and a truncated form of E2F1 RNA, respectively [29].
• By using RNase H to specifically fragment the IGF-II transcripts into 3' and 5' fragments, we found that the RNAs vary in size due to differences in the 5' end but not the 3' end [30].
• Oligonucleotide-directed RNase H digestion indicated regions of 7SK RNA capable of base pairing with other nucleic acids [31].
• The RNase H domain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase was released from recombinant DHFR-RNase H fusion protein by the action of HIV-1 protease and crystallized as large trigonal prisms that diffract x-rays to at least 2.4-A resolution [32].

Analytical, diagnostic and therapeutic context of RNASEH1

• Molecular cloning and expression of cDNA for human RNase H [33].
• The human RNASEH1 and mouse Rnaseh1 cDNAs were used to isolate BAC genomic clones that were used as probes for fluorescence in situ hybridization [16].
• RNase H digestion coupled with Northern blotting indicates that the two early crosslinks generated with P120-4SU+2 contain the U11 small nuclear RNA [34].
• Confirmation of the hierarchical folding of RNase H: a protein engineering study [35].
• Gel mobility shift assays with purified U1 snRNP and oligonucleotide-directed RNase H cleavage experiments support the idea that the intronic mutations destabilize a stem-loop structure that sequesters the 5' splice site downstream of exon 10 in tau pre-mRNA, leading to increases in U1 snRNP binding and in splicing between exon 10 and exon 11 [36].

References