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.
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, N2-(ADP-ribos-1-yl)-2'-deoxyguanosine (N2-ADPR-dG) in DNA. Here, we examined the involvement of the nucleotide excision repair (NER) system in the removal of N2-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 N2-ADPR-dG showed that E. coli UvrAB proteins bound to the N2-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 N2-ADPR-dGs in vitro. Finally, we introduced plasmids containing N2-ADPR-dGs into E. coli and human cells. N2-ADPR-adducted plasmids replicated l0 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 N2-ADPR-dG in both E. coli and human cells.
Kawanishi
Masanobu
M
Environmental Genetics Laboratory, Frontier Science Innovation Center, Osaka Prefecture University, 1-2 Gakuen-cho, Sakai, Osaka 599-8570, Japan.
Matsukawa
Kazuki
K
Kuraoka
Isao
I
Takamura-Enya
Takeji
T
Totsuka
Yukari
Y
Matsumoto
Yasuko
Y
Watanabe
Masahiko
M
Zou
Yue
Y
Tanaka
Kiyoji
K
Sugimura
Takashi
T
Wakabayashi
Keiji
K
Yagi
Takashi
T
D036002:ADP Ribose Transferases; D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000494:pharmacology*; D002080:Butterflies; D002384:Catalysis; D002460:Cell Line; D018736:DNA Adducts / Q000187:drug effects* / Q000378:metabolism; D004260:DNA Repair; D004261:DNA Replication / Q000187:drug effects; D004706:Endodeoxyribonucleases / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D006801:Humans; D019476:Insect Proteins / Q000494:pharmacology*; D015394:Molecular Structure; D009154:Mutation / Q000235:genetics; D009711:Nucleotides / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D051760:Xeroderma Pigmentosum Group A Protein / Q000378:metabolism
D051017:Apoptosis Regulatory Proteins; D018736:DNA Adducts; D029968:Escherichia coli Proteins; D019476:Insect Proteins; D009711:Nucleotides; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein; C113557:pierisin protein, insect; D000244:Adenosine Diphosphate; D036002:ADP Ribose Transferases; D004706:Endodeoxyribonucleases; C036385:endodeoxyribonuclease uvrABC