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Literature summary for 3.1.26.4 extracted from

  • Al-Behadili, A.; Uhler, J.P.; Berglund, A.K.; Peter, B.; Doimo, M.; Reyes, A.; Wanrooij, S.; Zeviani, M.; Falkenberg, M.
    A two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL (2018), Nucleic Acids Res., 46, 9471-9483 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 3.1.26.4

Cloned(Commentary)

Cloned (Comment) Organism
recombinant expression of C-terminally His6-tagged wild-type and mutant enzyme, without the predicted mitochondrial targeting sequence (residues 1-26), in Spodoptera frugiperda Sf9 insect cells using the baculovirus expression system Homo sapiens

Protein Variants

Protein Variants Comment Organism
A185V site-directed mutagenesis, reconstruction of a disease causing mutation in RNaseH1 that impairs primer processing. Ala185 is adjacent to the catalytically essential Glu186, which coordinates a catalytic Mg2+ ion and forms part of a hydrophobic pocket that mediates the stabilising interactions in the active site region. The A185V mutation causes a steric clash in this pocket Homo sapiens
additional information the potential structural/functional consequences of the V142I and A185V mutations are assessed by looking at the crystal structure of wild type RNase H1, PDB ID 2QK9. Both residues are located near the active site. The disease causing mutations disrupt the conformational stability of RNase H1. RNase H1 mutations impair primer removal at OriL in vivo Homo sapiens
V142I site-directed mutagenesis, reconstruction of a disease causing mutation in RNaseH1 that impairs primer processing. Val142 stabilises the hydrophobic interface between helix alpha1 and sheet beta1. This interface is critical for proper alignment of Asp145 and Glu186 in catalysis which is likely disturbed in the V142I variant Homo sapiens

Localization

Localization Comment Organism GeneOntology No. Textmining
mitochondrion
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 Homo sapiens 5739
-

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ required Homo sapiens

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
additional information Homo sapiens reconstitution of the replication cycle of L-strand synthesis in vitro using recombinant mitochondrial proteins and model OriL substrates: the process begins with initiation of DNA replication at OriL and ends with primer removal and ligation. RNase H1 partially removes the primer, leaving behind the last one to three ribonucleotides. These 5'-end ribonucleotides disturb ligation and are removed by Flap endonuclease 1 (FEN1) ?
-
 ?

Organism

Organism UniProt Comment Textmining
Homo sapiens O60930
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant C-terminally His6-tagged wild-type and mutant enzyme, without the predicted mitochondrial targeting sequence (residues 1-26), from insect Sf9 insect cells by nickel affinity and heparin affinity chromatography Homo sapiens

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
additional information reconstitution of the replication cycle of L-strand synthesis in vitro using recombinant mitochondrial proteins and model OriL substrates: the process begins with initiation of DNA replication at OriL and ends with primer removal and ligation. RNase H1 partially removes the primer, leaving behind the last one to three ribonucleotides. These 5'-end ribonucleotides disturb ligation and are removed by Flap endonuclease 1 (FEN1) Homo sapiens ?
-
 ?
additional information RNase H1 cleaves RNA in RNA-DNA hybrids to generate free 3'-OH and 5'-phosphate groups. The enzyme requires substrates containing at least four ribonucleotides to be active. RNase H1 cuts only between ribonucleotides, leaving at least 2 ribonucleotides attached to the 5'-end of the DNA. Nuclease activity reactions are performed on an 80 nt DNA template annealed to a 3' end labelled 52 nt chimeric oligonucleotide that contains 26 ribonucleotides followed by 26 deoxyribonucleotides (26RNA:26DNA) Homo sapiens ?
-
 ?

Synonyms

Synonyms Comment Organism
ribonulease H1
-
 Homo sapiens
RNase H1
-
 Homo sapiens

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
 assay at Homo sapiens

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
 assay at Homo sapiens

General Information

General Information Comment Organism
malfunction pathogenic consequences of disease causing mutations in RNase H1, overview. Loss of RNase H1 leads to primer retention at both OriH and OriL. RNase H1 mutations associated with mtDNA replication defects are identified in patients with mitochondrial encephalomyopathy. In vitro, the mutant proteins V142I, A185V, and R157stop have reduced activity on RNA-DNA hybrids. Analysis of patient samples show lower mtDNA levels and increased replication stalling. The disease causing mutations disrupt the conformational stability of RNase H1. RNase H1 mutations impair primer removal at OriL in vivo. The potential structural/functional consequences of the V142I and A185V mutations are assessed by looking at the crystal structure of wild-type RNase H1, PDB ID 2QK9. Both residues are located near the active site Homo sapiens
metabolism a two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL Homo sapiens
additional information Ala185 is adjacent to the catalytically essential Glu186, which coordinates a catalytic Mg2+ ion and forms part of a hydrophobic pocket that mediates the stabilising interactions in the active site region Homo sapiens
physiological function the role of Ribonuclease H1 (RNase H1) during primer removal and ligation at the mitochondrial origin of light-strand DNA synthesis (OriL) is a key step in mitochondrial DNA maintenance. FEN1 or an enzyme with FEN1-like activity is required for the last step of L-strand maturation before ligation. RNase H1 alone is insufficient for maturation of the nascent L-strand during DNA synthesis, the FEN1-like activity together with RNase H1 is needed for efficient ligation at OriL. But FEN1 is not able to substitute for RNase H1 during primer removal and L-strand maturation Homo sapiens