Related Experiment Video

Updated: Jun 23, 2026

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images
09:42

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

Published on: September 7, 2017

Modulation of the activity of methyl binding domain protein 4 (MBD4/MED1) while processing iododeoxyuridine generated

Mohammad Azhar Aziz1, Jane E Schupp, Timothy J Kinsella

  • 1Department of Radiation Oncology, University Hospitals Case Medical Center and the Case Integrative Cancer Biology Program, Case Western Reserve University, Cleveland, OH, USA.

Cancer Biology & Therapy
|April 28, 2009
PubMed

Insights

DNA glycosylases, including thymine DNA glycosylase (TDG) and methyl binding domain protein 4 (MBD4/MED1), repair chemotherapy-induced DNA damage. These enzymes specifically target G:IU mispairs, crucial for maintaining genomic integrity.

Area of Science:

  • Molecular Biology
  • Genomics
  • Biochemistry

Background:

  • DNA glycosylases are critical for genomic integrity, removing damaged DNA bases.
  • Chemotherapy and radiosensitizing drugs can introduce DNA base mispairs, creating a substrate for DNA glycosylases.
  • Iododeoxyuridine (IUdR) is a radiosensitizer that generates specific DNA mispairs.

Purpose of the Study:

  • To investigate the enzymatic activity of DNA glycosylases on mispairs generated by the radiosensitizer iododeoxyuridine (IUdR).
  • To characterize the substrate specificity of thymine DNA glycosylase (TDG), methyl binding domain protein 4 (MBD4/MED1), and uracil DNA glycosylase (UDG) towards IUdR-induced mispairs.
  • To determine the influence of DNA methylation on the activity of MBD4/MED1 and TDG on G:IU mispairs.

Main Methods:

  • Utilized a non-radioactive fluorescent dye-based in vitro glycosylase assay.
  • Quantitatively measured enzymatic activities of DNA glycosylases on G:IU and A:IU mispairs.
  • Assessed the impact of methylated CpG contexts on enzyme activity.

Main Results:

  • Thymine DNA glycosylase (TDG) and MBD4/MED1 demonstrated activity on G:IU mispairs but not A:IU mispairs.
  • UDG showed no activity on the tested IUdR-generated mispairs.
  • The methyl binding domain of MBD4/MED1 inhibited its own activity and glycosylase domain activity on G:IU mispairs within methylated CpG sites, but did not inhibit TDG.

Conclusions:

  • TDG and MBD4/MED1 are functionally complementary in repairing G:IU mispairs, contributing to genomic stability.
  • DNA methylation can modulate MBD4/MED1 activity on G:IU mispairs, highlighting context-dependent repair mechanisms.
  • UDG is not involved in the repair of IUdR-induced G:IU or A:IU mispairs.

More Related Videos

Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein11:01

Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein

Published on: March 31, 2010

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions08:14

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

Published on: July 17, 2019

Related Concept Videos

Mismatch Repair01:20

Mismatch Repair

Organisms are capable of detecting and fixing nucleotide mismatches that occur during DNA replication. This sophisticated process requires identifying the new strand and replacing the erroneous bases with correct nucleotides. Mismatch repair is coordinated by many proteins in both prokaryotes and eukaryotes.
The Mutator Protein Family Plays a Key Role in DNA Mismatch Repair
The human genome has more than 3 billion base pairs of DNA per cell. Prior to cell division, that vast amount of genetic...
Mismatch Repair01:36

Mismatch Repair

Overview
Mismatch Repair01:36

Mismatch Repair

Overview
Spontaneous and Induced Mutations01:30

Spontaneous and Induced Mutations

Spontaneous mutations arise infrequently during DNA replication due to errors in the process. A key factor behind these errors is tautomeric shifts in nitrogenous bases, where bases transition from keto to enol forms or amino to imino forms. This shift can alter base-pairing rules, leading to mutations. Additionally, reactive oxygen species (ROS) arising from aerobic metabolism can damage DNA, resulting in depurination (loss of a purine base) or depyrimidination (loss of a pyrimidine base).
Abnormal Proliferation02:23

Abnormal Proliferation

Under normal conditions, most adult cells remain in a non-proliferative state unless stimulated by internal or external factors to replace lost cells. Abnormal cell proliferation is a condition in which the cell's growth exceeds and is uncoordinated with normal cells. In such situations, cell division persists in the same excessive manner even after cessation of the stimuli, leading to persistent tumors. The tumor arises from the damaged cells that replicate to pass the damage to the daughter...
Proofreading01:43

Proofreading

Synthesis of new DNA molecules starts when DNA polymerase links nucleotides together in a sequence that is complementary to the template DNA strand. DNA polymerase has a higher affinity for the correct base to ensure fidelity in DNA replication. The DNA polymerase furthermore proofreads during replication, using an exonuclease domain that cuts off incorrect nucleotides from the nascent DNA strand.Errors during Replication Are Corrected by the DNA Polymerase EnzymeGenomic DNA is synthesized in...