A DNA site that has no base is called an AP site (APurinic/apyrimidinc site). AP sites arise from both spontaneous depurination/depyrimidination (about 10,000 per cell per day) and removal of a damaged base in the base excision repair (BER) pathway as an intermediate. AP sites are among the major DNA lesions and, if not repaired, inhibit replication and transcription or induce deletional mutations. AP endonuclease is an enzyme that recognizes AP sites and then excises the DNA strand at the site. The resulting nick in DNA is resynthesized and the repair is completed. The accumulation of such damage in DNA is thought to be related to aging, but this is still uncertain. In order to examine the relationship between DNA damage accumulation and aging, we are studying the DNA repair system in the worm C. elegans. C. elegans is very useful for the study of lifespan.
In this study, we focused on the apn-1 gene, which is a putative AP endonuclease in C. elegans. First, we cloned the apn-1 gene from a C. elegans cDNA library and purified APN-1 protein expressed as a GST-fusion protein in E. coli. The purified APN-1 protein showed excision activity toward AP sites in vitro. Second, we used an E. coli mutant that is deficient in AP endonucleases and therefore hypersensitive to DNA-damaging agents that produce AP sites. The E. coli mutant was rescued by expression of C. elegans APN-1. Third, we inactivated the apn-1 gene by feeding RNAi and now we are studying how this affects worms.