Site-directed mutagenesis of the human DNA repair enzyme HAP1: identification of residues important for AP endonuclease and RNase H activity
| Autor: | Gil Barzilay, Lisa J. Walker, Craig N. Robson, Ian D. Hickson |
|---|---|
| Rok vydání: | 1995 |
| Předmět: |
DNA Repair
Carbon-Oxygen Lyases Molecular Sequence Data Lyases AP endonuclease chemistry.chemical_compound DNA-(Apurinic or Apyrimidinic Site) Lyase Escherichia coli Genetics Animals Humans AP site Amino Acid Sequence Site-directed mutagenesis Exonuclease III Nuclease Base Sequence biology Escherichia coli Proteins Nuclear Proteins Ribonuclease Pancreatic DNA-(apurinic or apyrimidinic site) lyase Molecular biology Deoxyribonuclease IV (Phage T4-Induced) Enzyme Activation DNA/RNA non-specific endonuclease chemistry Biochemistry Mutagenesis Site-Directed biology.protein Drosophila Sequence Alignment DNA |
| Zdroj: | Nucleic Acids Research. 23:1544-1550 |
| ISSN: | 1362-4962 0305-1048 |
| DOI: | 10.1093/nar/23.9.1544 |
| Popis: | HAP1 protein, the major apurinic/apyrimidinic (AP) endonuclease in human cells, is a member of a homologous family of multifunctional DNA repair enzymes including the Escherichia coli exonuclease III and Drosophila Rrp1 proteins. The most extensively characterised member of this family, exonuclease III, exhibits both DNA- and RNA-specific nuclease activities. Here, we show that the RNase H activity characteristic of exonuclease III has been conserved in the human homologue, although the products resulting from RNA cleavage are dissimilar. To identify residues important for enzymatic activity, five mutant HAP1 proteins containing single amino acid substitutions were purified and analysed in vitro. The substitutions were made at sites of conserved amino acids and targeted either acidic or histidine residues because of their known participation in the active sites of hydrolytic nucleases. One of the mutant proteins (replacement of Asp-219 by alanine) showed a markedly reduced enzymatic activity, consistent with a greatly diminished capacity to bind DNA and RNA. In contrast, replacement of Asp-90, Asp-308 or Glu-96 by alanine led to a reduction in enzymatic activity without significantly compromising nucleic acid binding. Replacement of His-255 by alanine led to only a very small reduction in enzymatic activity. Our data are consistent with the presence of a single catalytic active site for the DNA- and RNA-specific nuclease activities of the HAP1 protein. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|