Related Experiment Video

Updated: Oct 3, 2026

Atomic Force Microscopy Investigations of DNA Lesion Recognition in Nucleotide Excision Repair
10:59

Atomic Force Microscopy Investigations of DNA Lesion Recognition in Nucleotide Excision Repair

Published on: May 24, 2017

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.

Nucleic Acids Research
|November 3, 2001
PubMed

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.

More Related Videos

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis11:08

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

Published on: June 19, 2018

Related Concept Videos

Nucleotide Excision Repair01:46

Nucleotide Excision Repair

Exposure to mutagens can damage DNA and result in bulky lesions that distort the double-helix structure or impede proper transcription. Damaged DNA can be detected and repaired in a process called nucleotide excision repair (NER). NER employs a set of specialized proteins that first scan DNA to detect a damaged region. Next, NER proteins separate the strands and excise the damaged area. Finally, they coordinate the replacement with new, matching nucleotides.DNA distortion and damageCells are...
Nucleotide Excision Repair01:38

Nucleotide Excision Repair

DNA Distortion and Damage
Cells are regularly exposed to mutagens—factors in the environment that can damage DNA and generate mutations. UV radiation is one of the most common mutagens and is estimated to introduce a significant number of changes in DNA. These include bends or kinks in the structure, which can block DNA replication or transcription. If these errors are not fixed, the damage can cause mutations, which in turn can result in cancer or disease depending on which sequences are...
Nucleotide Excision Repair01:46

Nucleotide Excision Repair

Exposure to mutagens can damage DNA and result in bulky lesions that distort the double-helix structure or impede proper transcription. Damaged DNA can be detected and repaired in a process called nucleotide excision repair (NER). NER employs a set of specialized proteins that first scan DNA to detect a damaged region. Next, NER proteins separate the strands and excise the damaged area. Finally, they coordinate the replacement with new, matching nucleotides.DNA distortion and damageCells are...
Base Excision Repair01:54

Base Excision Repair

One of the common DNA damages is the chemical alteration of single bases by alkylation, oxidation, or deamination. The altered bases cause mispairing and strand breakage during replication. This type of damage causes minimal change to the DNA double helix structure and can be repaired by the base excision repair (BER) pathways. BER corrects damaged DNA sequences by removing the damaged base and restoring the original base sequence using the complementary strand as a template.
The first step of...
Base Excision Repair01:54

Base Excision Repair

One of the common DNA damages is the chemical alteration of single bases by alkylation, oxidation, or deamination. The altered bases cause mispairing and strand breakage during replication. This type of damage causes minimal change to the DNA double helix structure and can be repaired by the base excision repair (BER) pathways. BER corrects damaged DNA sequences by removing the damaged base and restoring the original base sequence using the complementary strand as a template.
The first step of...
Homologous Recombination02:31

Homologous Recombination

The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...