Cloned (Comment) | Organism |
---|---|
gene rnh, DNA and amino acid sequence determination and analysis of the rnh genes from phages T4D, das13, NG163das13 and NG163, recombinant expression of N-terminally His6-tagged wild-type and mutant enzymes in Escherichia coli strain L21(DE3)/pLysE | Tequatrovirus T4 |
Protein Variants | Comment | Organism |
---|---|---|
C724A | site-directed mutagenesis | Tequatrovirus T4 |
L242I | naturally occuring mutation, the substitution does not affect the structure of RNase H and its role in providing the das-effect remains unclear | Tequatrovirus T4 |
V43I | naturally occuring mutation, the V43I substitution may lead to disposition of H4 helix, responsible for the interaction with the first base pairs of 5' end of branched DNA. These structural changes may affect unwinding of the first base pairs of gapped or nicked DNA generating a short flap and therefore may stabilize the DNA-enzyme complex | Tequatrovirus T4 |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | Tequatrovirus T4 | phage T4 RNase H shows 5'-3'exonuclease (EC 3.1.13.2) and flap endonuclease (EC 3.1.99.) activities on dsDNA. THe enzyme exhibits also endonucleolytic cleavage to 5'- phosphomonoester, EC 3.1.26.4 | ? | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Tequatrovirus T4 | P13319 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant N-terminally His6-tagged wild-type and mutant enzymes from Escherichia coli strain L21(DE3)/pLysE by nickel affinity chromatography, tag cleavage by TEV protease, dialysis, and again nickel affinity chromatography and dialysis | Tequatrovirus T4 |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | phage T4 RNase H shows 5'-3'exonuclease (EC 3.1.13.2) and flap endonuclease (EC 3.1.99.) activities on dsDNA. THe enzyme exhibits also endonucleolytic cleavage to 5'- phosphomonoester, EC 3.1.26.4 | Tequatrovirus T4 | ? | - |
? | |
additional information | synthetic DNA substrates are used | Tequatrovirus T4 | ? | - |
? |
Synonyms | Comment | Organism |
---|---|---|
ribonuclease H | - |
Tequatrovirus T4 |
RNase H | - |
Tequatrovirus T4 |
RNH | - |
Tequatrovirus T4 |
T4 RNase H | - |
Tequatrovirus T4 |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Tequatrovirus T4 |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Tequatrovirus T4 |
General Information | Comment | Organism |
---|---|---|
evolution | RNase H belongs to the eukaryotic Mre11/Rad50 (MR) and bacterial SbcCD complex family. The crystal structure of T4 RNase H shows a close homology to flap endonucleases of the FEN-1 family rather than RNase H family. FEN-1 is a family of structure-specific 5'-nucleases, which is conserved from bacteriophage to humans | Tequatrovirus T4 |
malfunction | the V43I substitution increases the ratio between exonuclease (EC 3.1.13.2) and endonuclease (EC 3.1.99.) activities of RNase H whereas L242I substitution does not affect the nuclease activity of RNase H in vitro. The Das13 mutant of RNase H has substitutions of valine 43 and leucine 242 with isoleucines. Both mutations are necessary for the full das mutant effect in vivo. The V43I substitution may lead to disposition of H4 helix, responsible for the interaction with the first base pairs of 5' end of branched DNA. These structural changes may affect unwinding of the first base pairs of gapped or nicked DNA generating a short flap and therefore may stabilize the DNA-enzyme complex. The L242I substitution does not affect the structure of RNase H and its role in providing the das-effect remains unclear | Tequatrovirus T4 |
additional information | molecular modelling of the nuclease structure, and modeling of the function of flap endonucleases | Tequatrovirus T4 |
physiological function | RNase H is a 5'-nuclease required to remove RNA primers from lagging strand fragments during DNA replication. It forms the gp46/47 enzyme complex consists of a DNA-activated ATPase, an ssDNA endonuclease and a 3'-5' dsDNA exonuclease | Tequatrovirus T4 |