Abstract
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. To confirm that APN-1 does have an AP endonuclease activity, we first 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 indeed showed an excision activity toward AP sites in vitro. Then 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. Next we produced a rabbit polyclonal antibody against APN-1 protein. We also produced transformed C. elegans that is expressing APN-1-GFP fusion protein and now we are investigating when, where, and how APN-1 functions in C. elegans.
