Cloned (Comment) | Organism |
---|---|
expression of human RNase H1 in Escherichia coli | Homo sapiens |
expression of mutant enzymes in Escherichia coli | Halalkalibacterium halodurans |
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 | 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 | 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 | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
DNA-RNA hybrid + H2O | Homo sapiens | - |
DNA + 5'-phosphonucleotides | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Halalkalibacterium halodurans | Q9KEI9 | - |
- |
Homo sapiens | - |
- |
- |
Purification (Comment) | Organism |
---|---|
recombinant RNase H1 from Escherichia coli strain BL21(DE3) using affinity and ion exchange chromatography | Homo sapiens |
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 | 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 | 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 | Comment | Organism |
---|---|---|
RNase H | - |
Halalkalibacterium halodurans |
RNase H1 | - |
Homo sapiens |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
22 | - |
assay at | Homo sapiens |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.9 | - |
assay at | Homo sapiens |