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Literature summary for 3.1.13.2 extracted from

  • Kholod, N.; Sivogrivov, D.; Latypov, O.; Mayorov, S.; Kuznitsyn, R.; Kajava, A.V.; Shlyapnikov, M.; Granovsky, I.
    Single substitution in bacteriophage T4 RNase H alters the ratio between its exo- and endonuclease activities (2015), Mutat. Res., 781, 49-57 .
    View publication on PubMed
Search for more literature for 3.1.13.2

Cloned(Commentary)

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

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

Localization

Localization Comment Organism GeneOntology No. Textmining
membrane bound Tequatrovirus T4 16020
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Natural Substrates/ Products (Substrates)

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 ?
-
 ?

Organism

Organism UniProt Comment Textmining
Tequatrovirus T4 P13319
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-

Purification (Commentary)

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 and Products (Substrate)

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 Tequatrovirus T4 ?
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 ?
additional information synthetic DNA substrates are used Tequatrovirus T4 ?
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 ?

Synonyms

Synonyms Comment Organism
ribonuclease H
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 Tequatrovirus T4
RNase H
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 Tequatrovirus T4
RNH
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 Tequatrovirus T4
T4 RNase H
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 Tequatrovirus T4

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
 assay at Tequatrovirus T4

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
 assay at Tequatrovirus T4

General Information

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 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 consisting of a DNA-activated ATPase, an ssDNA endonuclease and a 3'-5' dsDNA exonuclease Tequatrovirus T4