Nucleic Acids Research, 1995, Vol. 23, No. 9 1544-1550
© 1995
MOLECULAR BIOLOGY |
Site-directed mutagenesis of the human DNA repair enzyme HAP1: identification of residues important for AP endonuclease and RNase H activity
Imperial Cancer Research Fund Laboratories, Institute of Molecular Medicine, University of Oxford John Radcliffe Hospital, Oxford 0X3 9DU, UK
*To whom correspondence should be addressed
Received December 19, 1994. Revised March 24, 1995. Accepted March 24, 1995.
HAP1 protein, the major apurinic/apyrimldinic (AP) endonuclease in human cells, Is a member of a homologous family of multifunctional DNA repair enzymes including the Escherichla coll exonuclease III and Drosophlla 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 histidlne residues because of their known participation in the active sites of hydroly-tic nucleases. One of the mutant proteins (replacement of Asp-219 by alanlne) 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.
+Present address: Department of Surgery, Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, UK
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