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Literature summary for 3.1.26.4 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)

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

Crystallization (Commentary)

Crystallization (Comment) Organism
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
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

Protein Variants Comment Organism
D132N site-directed mutagenesis, crystal structure analysis of the mutant bound to divalent metal ions Halalkalibacterium halodurans
D192N site-directed mutagenesis, crystal structure analysis of the mutant bound to divalent metal ions Halalkalibacterium halodurans
E188A site-directed mutagenesis, crystal structure analysis of the mutant bound to divalent metal ions Halalkalibacterium halodurans
K59A/K60A site-directed mutagenesis in the hybrid binding domain, the mutation abolishes dsRNA binding Homo sapiens
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
R32A/R33A site-directed mutagenesis in the hybrid binding domain outside of the interface, the mutation abolishes dsRNA binding Homo sapiens
R35A site-directed mutagenesis, the mutant shows a much lower specific activity than the wild-type enzyme Homo sapiens
R57A site-directed mutagenesis in the hybrid binding domain on the observed nucleic-acid interface, the mutation abolishes dsRNA binding Homo sapiens
R72A/K73A site-directed mutagenesis in the hybrid binding domain outside of the interface, the mutation abolishes dsRNA binding Homo sapiens
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
Y29F site-directed mutagenesis in the hybrid binding domain, the mutation abolishes dsRNA binding Homo sapiens

Metals/Ions

Metals/Ions Comment Organism Structure
Ca2+ binding structure Halalkalibacterium halodurans
Mg2+
-
Homo sapiens
Mg2+ binding structure Halalkalibacterium halodurans
Mn2+ binding structure Halalkalibacterium halodurans
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)

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

Organism

Organism UniProt Comment Textmining
Halalkalibacterium halodurans Q9KEI9
-
-
Homo sapiens
-
-
-

Purification (Commentary)

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

Reaction

Reaction Comment Organism Reaction ID
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

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
DNA-RNA hybrid + H2O
-
Halalkalibacterium halodurans ?
-
?
DNA-RNA hybrid + H2O
-
Homo sapiens DNA + 5'-phosphonucleotides
-
?
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

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

Synonyms

Synonyms Comment Organism
RNase H
-
Halalkalibacterium halodurans
RNase H1
-
Homo sapiens

Temperature Optimum [°C]

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

pH Optimum

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