Related Experiment Video
Updated: Mar 12, 2026
Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy
Published on: June 25, 2013
Mre11 Is Essential for the Removal of Lethal Topoisomerase 2 Covalent Cleavage Complexes
Nguyen Ngoc Hoa1, Tsubasa Shimizu1, Zhong Wei Zhou2
1Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshida Konoe, Sakyo-ku, Kyoto 606-8501, Japan.
Abstract:
The Mre11/Rad50/Nbs1 complex initiates double-strand break repair by homologous recombination (HR). Loss of Mre11 or its nuclease activity in mouse cells is known to cause genome aberrations and cellular senescence, although the molecular basis for this phenotype is not clear. To identify the origin of these defects, we characterized Mre11-deficient (MRE11-/-) and nuclease-deficient Mre11 (MRE11-/H129N) chicken DT40 and human lymphoblast cell lines. These cells exhibit increased spontaneous chromosomal DSBs and extreme sensitivity to topoisomerase 2 poisons. The defects in Mre11 compromise the repair of etoposide-induced Top2-DNA covalent complexes, and MRE11-/- and MRE11-/H129N cells accumulate high levels of Top2 covalent conjugates even in the absence of exogenous damage. We demonstrate that both the genome instability and mortality of MRE11-/- and MRE11-/H129N cells are significantly reversed by overexpression of Tdp2, an enzyme that eliminates covalent Top2 conjugates; thus, the essential role of Mre11 nuclease activity is likely to remove these lesions.
Insights
The Mre11/Rad50/Nbs1 complex is crucial for DNA repair. Loss of Mre11 nuclease activity leads to genome instability by failing to remove Top2-DNA complexes, a defect reversed by Tdp2.
Area of Science:
- DNA repair mechanisms
- Genome stability
- Homologous recombination
Background:
- The Mre11/Rad50/Nbs1 complex initiates double-strand break repair via homologous recombination (HR).
- Loss of Mre11 or its nuclease activity causes genome aberrations and senescence, but the molecular basis is unclear.
Purpose of the Study:
- To investigate the molecular basis of genome instability and mortality in Mre11-deficient cells.
- To determine the role of Mre11 nuclease activity in repairing Topoisomerase 2 (Top2) related DNA damage.
Main Methods:
- Characterization of Mre11-deficient (MRE11-/-) and nuclease-deficient Mre11 (MRE11-/H129N) chicken DT40 and human lymphoblast cell lines.
- Assessment of spontaneous chromosomal double-strand breaks (DSBs) and sensitivity to Top2 poisons.
- Evaluation of etoposide-induced Top2-DNA covalent complex repair and accumulation.
- Analysis of the effect of Tdp2 overexpression on Mre11-deficient cell phenotypes.
Main Results:
- MRE11-/- and MRE11-/H129N cells exhibit increased spontaneous DSBs and extreme sensitivity to Top2 poisons.
- These cells show compromised repair of etoposide-induced Top2-DNA covalent complexes and accumulate high levels of Top2 conjugates.
- Genome instability and mortality in MRE11-/- and MRE11-/H129N cells were significantly reversed by Tdp2 overexpression.
Conclusions:
- Mre11 nuclease activity is essential for removing Top2-DNA covalent conjugates.
- Failure to remove these lesions contributes to the genome instability and cell death observed in Mre11-deficient cells.
Related Concept Videos
02:02DNA Topoisomerases
Types and Mechanism of action
Topoisomerases are divided into two main types. ...
02:31Homologous Recombination
01:20Mismatch Repair
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...
01:36Mismatch Repair
01:36Mismatch Repair
02:53Conservative Site-specific Recombination and Phase Variation
The recognition sites for Cre recombinase called LoxP...