Reactivity of human apurinic/apyrimidinic endonuclease and Escherichia coli exonuclease III with bistranded abasic sites in DNA

Abstract

Several oxidative DNA-damaging agents, including ionizing radiation, can generate multiply damaged sites in DNA. Among the postulated lesions are those with abasic sites located in close proximity on opposite strands. The repair of an abasic site requires strand scission by a repair endonuclease such as human apurinic/apyrimidinic endonuclease (Ape) or exonuclease III in Escherichia coli. Therefore, a potential consequence of the 'repair' of bistranded abasic sites is the formation of double-strand breaks. To test this possibility and to investigate the influence of the relative distance between the two abasic sites and their orientation to each other, we prepared a series of oligonucleotide duplexes containing abasic sites at defined positions either directly opposite each other or separated by 1, 3, or 5 base pairs in the 5'- or 3'-direction. Analysis following Ape and exonuclease III treatment of these substrates indicated a variety of responses. In general, cleavage at abasic sites was slower in duplexes with paired lesions than in control duplexes with single lesions. Double-strand breaks were, however, readily generated in duplexes with abasic sites positioned 3' to each other. With the duplex containing abasic sites set 1 base pair apart, 5' to each other, both Ape and exonuclease III slowly cleaved the abasic site on one strand only and were unable to incise the other strand. With the duplex containing abasic sites set 3 base pairs apart, 5' to each other, Ape protein was unable to cleave either strand. These data suggest that closely positioned abasic sites could have several deleterious consequences in the cell. In addition, this approach has allowed us to map bases that make significant contact with the enzymes when acting on an abasic site on the opposite strand.