Human apurinic/apyrimidinic endonuclease is processive

Apurinic/apyrimidinic endonuclease (AP endo) is believed to play a critical role in repair of oxidative damage of DNA and is proposed to initiate repair of most abasic sites in the base excision repair pathway. AP endo makes a single nick 5' to an abasic site in double-stranded DNA. In this study, we investigated whether AP endo locates an abasic site through a processive or a distributive mechanism. We used a linear multi-abasic site substrate (concatemer), synthesized by ligating together identical 25-nucleotide monomeric units (25-mers). We first determined that the 25-mer monomer from which the concatemers were prepared was nicked by AP endo in a fashion similar to that of the previously published 49-mer substrate with a different sequence. Steady state parameters K(m)and k(cat) and single-turnover parameters for substrate binding were comparable to previously published values. Using the multi-abasic site concatemer, we demonstrated that AP endo was capable of cleaving approximately seven to eight abasic sites, traveling at least 200 nucleotides, before dissociating from its substrate. Thus, AP endo, like uracil DNA glycosylase, behaves in a quasi processive fashion. Processivity could be separated from catalysis, since processivity was maximal at 25 mM NaCl, while the rate of cleavage was maximal at 125 mM salt. In short, nicking activity was maximized close to physiological salt molarities while processivity was midrange at physiological salt concentrations. The latter is likely to be subject to tight regulation by small changes in ionic strength.