SITE-DIRECTED MUTAGENESIS OF THE HUMAN DNA-REPAIR ENZYME HAP1 - IDENTIFICATION OF RESIDUES IMPORTANT FOR AP ENDONUCLEASE AND RNASE-H ACTIVITY

HAP1 protein, the major apurinic/apyrimidinic (AP) endonuclease in hum an cells, is a member of a homologous family of multifunctional DNA re pair enzymes including the Escherichia coli exonuclease III and Drosop hila 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 th e products resulting from RNA cleavage are dissimilar. To identify res idues important for enzymatic activity, five mutant HAP1 proteins cont aining single amino acid substitutions were purified and analysed in v itro. The substitutions were made at sites of conserved amino acids an d 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 capac ity to bind DNA and RNA. In contrast, replacement of Asp-90, Asp-308 o r Glu-96 by alanine led to a reduction in enzymatic activity without s ignificantly 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 pr otein.