Abstract
Apurinic/apyrimidinic endonuclease plays an essential role in DNA base excision repair (BER). APE1 (aka APEX1, or Ref1), the mammalian AP endonuclease, is also known to have gene regulation functions as a redox-enhancing facotr or as the acetylation-mediated co-repressor. APE1 null embryos die just after blastocyst formation, and there has been no report of isolation of its mouse embryonic fibroblast (MEF) cells. To understand APE1's in vivo role more precisely, a conditional ko MEFs were isolated in which APE1 could be removed by excising the APE1 gene with the Cre recombinase, and the fate of the cells without APE1 was analyzed.
The cre gene was introduced by a direct nuclear microinjection. The MEF cells became apoptotic within 12 h after the cre injection, and almost all the cells were in a late apoptotic stage in 24 h. The MEF cells were normal when the cre as well as the wild-type APE1 cDNA were co-injected, indicating that APE1 is essential even in cultured cells. A "complementation test" was carried out where the cre gene was co-injected with missensed APE1 cDNA, each of which lacks a particular function of APE1. Based on this experiment, we concluded that the AP endonuclease activity as well as the aceytlation-mediated co-repressor functions are absolutely required for the cells.
