Repair of triplex-directed DNA alkylation by nucleotide excision repair.
Triplex-forming oligonucleotides (TFOs) are being investigated as highly specific DNA binding agents to inhibit the expression of clinically relevant genes. So far, they have been shown to inhibit transcription from the HER-2/neu gene in vitro, whereas their use in vivo has been studied to a limited extent. This study uses a TFO-chlorambucil (chl) conjugate capable of forming site-specific covalent guanine adducts within the HER-2/neu promoter. We demonstrate that nucleotide excision repair (NER) represents a mechanism of cellular resistance to TFO-directed DNA alkylation. In vitro repair assays demonstrate that triplex-directed chl-guanine adducts are substrates for repair by NER competent cell extracts but not XP12BE cell extracts deficient in NER. The degree of repair is estimated by a ligation-mediated polymerase chain reaction with a pre-formed triplex in a plasmid transfected into repair competent cells, indicating that approximately 25% of the guanine adducts are removed after 24 h. These data indicate that guanine adducts from TFO-directed alkylation are a substrate for NER and that DNA repair is a significant barrier to the intracellular persistence of target gene binding by TFOs.
Ziemba
A
A
Arizona Cancer Center, University of Arizona, 1515 North Campbell Avenue, Tucson, AZ 85724-5024, USA.
Derosier
L C
LC
Methvin
R
R
Song
C Y
CY
Clary
E
E
Kahn
W
W
Milesi
D
D
Gorn
V
V
Reed
M
M
Ebbinghaus
S
S
D000478:Alkylation; D004247:DNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018736:DNA Adducts / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004249:DNA Damage / Q000235:genetics*; D011088:DNA Ligases / Q000378:metabolism; D004260:DNA Repair / Q000235:genetics*; D018734:Genes, erbB-2 / Q000235:genetics; D019791:Guanidine / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010880:Piperidines / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D013379:Substrate Specificity; D014162:Transfection
D018736:DNA Adducts; D009838:Oligodeoxyribonucleotides; D010880:Piperidines; C051802:triplex DNA; C032727:piperidine; D004247:DNA; D011088:DNA Ligases; D019791:Guanidine