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Literature summary extracted from

  • Nowotny, M.; Yang, W.
    Stepwise analyses of metal ions in RNase H catalysis from substrate destabilization to product release (2006), EMBO J., 25, 1924-1933.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.1.26.4 expression of human RNase H1 in Escherichia coli Homo sapiens
3.1.26.4 expression of mutant enzymes in Escherichia coli Halalkalibacterium halodurans

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
3.1.26.4 purified recombinant mutant enzymes, with bound metal ions, sitting drop vapour diffusion method, 4°C, 25-30% 2-metyl-2,4-pentanediol, as precipitant, 0.25 M ammonium acetate or 0.2 M NaCl, sodium citrateat pH 5.6, Tris at pH 7.0, or HEPES at pH 7.5, Microseeding with mutant D132N, nick phosphorylation and 20% ethanol at pH 7.0 for mutant E188A, soaking of crystals in solution containing Mg2+, Mn2+, or Ca2+ for 4-24 h, X-ray diffraction structure determination and analysis at 1.5-2.2 A resolution Halalkalibacterium halodurans
3.1.26.4 the enzyme's hybrid binding domain complexed with a 12 bp RNA-DNA hybrid and an 6 bp RNA-DNA hybrid, molecular replacement, hanging drop vapour diffusion method, 21°C, 6 bp complex crystals are obtained with the well solution containing 10% PEG 3350, 0.2 M NaCl, 0.1 M Tris, pH 8.5, 12 bp complex crystals are obtained with 1.2 M NaCl and 0.1 M HEPES, pH 7.5, X-ray diffraction structure determination and anaylsis at 2.7-2.8 A and 2.1-2.2 A resolution, respectively Homo sapiens

Protein Variants

EC Number Protein Variants Comment Organism
3.1.26.4 D132N site-directed mutagenesis, crystal structure analysis of the mutant bound to divalent metal ions Halalkalibacterium halodurans
3.1.26.4 D192N site-directed mutagenesis, crystal structure analysis of the mutant bound to divalent metal ions Halalkalibacterium halodurans
3.1.26.4 E188A site-directed mutagenesis, crystal structure analysis of the mutant bound to divalent metal ions Halalkalibacterium halodurans
3.1.26.4 K59A/K60A site-directed mutagenesis in the hybrid binding domain, the mutation abolishes dsRNA binding Homo sapiens
3.1.26.4 additional information full-length enzymes with defective HBDs indicates that this domain dramatically enhances both the specific activity and processivity of RNase H1 Homo sapiens
3.1.26.4 R32A/R33A site-directed mutagenesis in the hybrid binding domain outside of the interface, the mutation abolishes dsRNA binding Homo sapiens
3.1.26.4 R35A site-directed mutagenesis, the mutant shows a much lower specific activity than the wild-type enzyme Homo sapiens
3.1.26.4 R57A site-directed mutagenesis in the hybrid binding domain on the observed nucleic-acid interface, the mutation abolishes dsRNA binding Homo sapiens
3.1.26.4 R72A/K73A site-directed mutagenesis in the hybrid binding domain outside of the interface, the mutation abolishes dsRNA binding Homo sapiens
3.1.26.4 W43A site-directed mutagenesis in the hybrid binding domain on the observed nucleic-acid interface, the mutation abolishes dsRNA binding, the mutant shows a much lower specific activity than the wild-type enzyme Homo sapiens
3.1.26.4 Y29F site-directed mutagenesis in the hybrid binding domain, the mutation abolishes dsRNA binding Homo sapiens

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
3.1.26.4 Ca2+ binding structure Halalkalibacterium halodurans
3.1.26.4 Mg2+
-
 Homo sapiens
3.1.26.4 Mg2+ binding structure Halalkalibacterium halodurans
3.1.26.4 Mn2+ binding structure Halalkalibacterium halodurans
3.1.26.4 additional information the enzyme performs a two-metal catalysis, with metal A activating the nucleophile and metal B stabilizing the transition state, mechanism and structures, overview Halalkalibacterium halodurans

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
3.1.26.4 DNA-RNA hybrid + H2O Homo sapiens
-
 DNA + 5'-phosphonucleotides
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
3.1.26.4 Halalkalibacterium halodurans Q9KEI9
-
-
3.1.26.4 Homo sapiens
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.1.26.4 recombinant RNase H1 from Escherichia coli strain BL21(DE3) using affinity and ion exchange chromatography Homo sapiens

Reaction

EC Number Reaction Comment Organism Reaction ID
3.1.26.4 Endonucleolytic cleavage to a 5'-phosphomonoester the enzyme performs a two-metal catalysis, with metal A activating the nucleophile and metal B stabilizing the transition state, mechanism and structures, overview Halalkalibacterium halodurans
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.1.26.4 DNA-RNA hybrid + H2O
-
 Halalkalibacterium halodurans ?
-
 ?
3.1.26.4 DNA-RNA hybrid + H2O
-
 Homo sapiens DNA + 5'-phosphonucleotides
-
 ?
3.1.26.4 DNA-RNA hybrid + H2O poly-rA/poly-dT substrate, RNase H1 contains an N-terminal domain termed dsRHbd or hybrid binding domain for binding both dsRNA and RNA/DNA hybrid, the RNA strand is recognized by a protein loop, which forms hydrogen bonds with the 2'-OH groups, substrate recognition and binding structure, residues, Y29, R32, R33, W43, R57, K59, K60, R72, and K73 are involved, overview Homo sapiens DNA + 5'-phosphonucleotides determination of reaction products with less than 20-nucleotides ?

Subunits

EC Number Subunits Comment Organism
3.1.26.4 More modeling and analysis of mutant enzymes structures with bound DNA/RNA hybrid substrates, overview Halalkalibacterium halodurans
3.1.26.4 More RNase H1 contains an N-terminal domain known as dsRHbd for binding both dsRNA and RNA/DNA hybrid Homo sapiens

Synonyms

EC Number Synonyms Comment Organism
3.1.26.4 RNase H
-
 Halalkalibacterium halodurans
3.1.26.4 RNase H1
-
 Homo sapiens

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.1.26.4 22
-
 assay at Homo sapiens

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.1.26.4 7.9
-
 assay at Homo sapiens