Molecular Evidence of the Involvement of the Nucleotide Excision Repair (NER) System in the Repair of the Mono(ADP-Ribosyl)ated DNA Adduct Produced by Pierisin-1, an Apoptosis-Inducing Protein from the Cabbage Butterfly

摘要

Pierisin-1 is a potent apoptosis-inducing protein found in the pupal extract of the cabbage white butterfly. Pierisin-1 catalyzes the mono(ADP-ribosyl)ation of the 2'-deoxyguanosine residue and produces a bulky adduct, N~2-(ADP-ribos-l-yl)-2'-deoxyguanosine (N~2-ADPR-dG) in DNA. Here, we examined the involvement of the nucleotide excision repair (NER) system in the removal of N~2-ADPR-dG in Escherichia coli (E. coli) and human cells. The results of mobility shift gel electrophoresis assays using a 50-mer oligodeoxynucleotide containing a single N~2-ADPR-dG showed that E. coli UvrAB proteins bound to the N~2-ADPR-dG in vitro. Incubation of the adducted oligodeoxynucleotides with UvrABC resulted in the incision of the oligonucleotides in vitro. The results of filter binding and gel mobility shift assays using human XPA protein showed that XPA bound to DNA containing N~2-ADPR-dGs in vitro. Finally, we introduced plasmids containing N~2-ADPR-dGs into E. coli and human cells. N~2-ADPR-adducted plasmids replicated 10 times and 20 times less efficiently in NER-deficient E. coli and human cells than in their wild-type counterparts, respectively. More mutations were induced in the plasmid propagated in NER-deficient cells than that in wild-type human cells. These results indicate the involvement of the NER system in the repair of N~2-ADPR-dG in both E. coli and human cells.