Characterization of amino acid substitutions that severely alter the DNA repair functions of Escherichia coli endonuclease IV

Escherichia coli endo IV is a bifunctional DNA repair protein, i.e., posses sing both apurinic/ apyrimidinic (AP) endonuclease and 3'-diesterase activi ties. The former activity cleaves AP sites, whereas the latter one removes a variety of 3'-blocking groups present at single-strand breaks in damaged DNA. However, the precise reaction mechanism by which endo IV cleaves DNA l esions is unknown. To probe this mechanism, we have identified eight amino acid substitutions that alter endo IV function in vivo. Seven of these muta nt proteins are variably expressed in E. coli and, when purified, show a 10 -60-fold reduction in both AP endonuclease and 3'-diesterase activities. Th e most severe defect was observed with the one remaining mutant (E145G) tha t showed normal protein expression. This mutant has lost the ability to bin d double-stranded DNA and showed a dramatic 150-fold reduction in enzymatic activities. We conclude that the AP endonuclease and the 3'-diesterase act ivities of endo IV are associated with a single active site, that is perhap s remote from the DNA binding domain.