Related Experiment Videos
Repair of triplex-directed DNA alkylation by nucleotide excision repair
A Ziemba1, L C Derosier, R Methvin
1Arizona Cancer Center, University of Arizona, 1515 North Campbell Avenue, Tucson, AZ 85724-5024, USA.
Abstract:
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.
Insights
Triplex-forming oligonucleotides (TFOs) show promise for gene silencing. However, cellular DNA repair mechanisms, specifically nucleotide excision repair (NER), can remove TFO-induced DNA damage, limiting their effectiveness in vivo.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Triplex-forming oligonucleotides (TFOs) are investigated for specific gene expression inhibition.
- Previous studies showed TFOs inhibit HER-2/neu gene transcription in vitro.
- Limited in vivo studies exist for TFO applications.
Purpose of the Study:
- To investigate the role of nucleotide excision repair (NER) in cellular resistance to TFO-directed DNA alkylation.
- To assess the repair of TFO-chlorambucil (chl) guanine adducts by NER.
- To understand DNA repair as a barrier to TFO efficacy in vivo.
Main Methods:
- Utilized a TFO-chlorambucil (chl) conjugate for site-specific guanine adduct formation in the HER-2/neu promoter.
- Performed in vitro repair assays using NER-competent and NER-deficient cell extracts.
- Quantified adduct repair using ligation-mediated polymerase chain reaction (LM-PCR) in transfected cells.
Main Results:
- TFO-directed chl-guanine adducts are substrates for repair by NER-competent cell extracts.
- Adducts were not repaired by NER-deficient XP12BE cell extracts.
- Approximately 25% of guanine adducts were removed by NER within 24 hours in repair-competent cells.
Conclusions:
- Nucleotide excision repair (NER) is a significant mechanism of cellular resistance to TFO-directed DNA alkylation.
- TFO-induced guanine adducts are substrates for NER.
- DNA repair pathways represent a substantial barrier to the intracellular persistence and efficacy of TFOs for gene targeting.