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Mre11 and Rad50 from Pyrococcus furiosus: cloning and biochemical characterization reveal an evolutionarily conserved
K P Hopfner1, A Karcher, D Shin
1Department of Molecular Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Abstract:
The processing of DNA double-strand breaks is a critical event in nucleic acid metabolism. This is evidenced by the severity of phenotypes associated with deficiencies in this process in multiple organisms. The core component involved in double-strand break repair in eukaryotic cells is the Mre11-Rad50 protein complex, which includes a third protein, p95, in humans and Xrs2 in yeasts. Homologues of Mre11 and Rad50 have been identified in all kingdoms of life, while the Nbs1 protein family is found only in eukaryotes. In eukaryotes the Mre11-Rad50 complex has nuclease activity that is modulated by the addition of ATP. We have isolated the Mre11 and Rad50 homologues from the thermophilic archaeon Pyrococcus furiosus and demonstrate that the two proteins exist in a large, heat-stable complex that possesses single-strand endonuclease activity and ATP-dependent double-strand-specific exonuclease activity. These findings verify the identification of the P. furiosus Rad50 and Mre11 homologues and demonstrate that functional homologues with similar biochemical properties exist in all kingdoms of life.
Insights
DNA double-strand break repair is vital. Researchers found heat-stable Mre11-Rad50 archaeal complex with nuclease activity, confirming its presence across all life kingdoms.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- DNA double-strand breaks are critical nucleic acid events.
- Deficiencies in DNA repair lead to severe phenotypes.
- The Mre11-Rad50 complex is central to eukaryotic double-strand break repair.
Purpose of the Study:
- To isolate and characterize Mre11 and Rad50 homologues from Pyrococcus furiosus.
- To investigate the biochemical properties of the archaeal Mre11-Rad50 complex.
Main Methods:
- Isolation of Mre11 and Rad50 proteins from Pyrococcus furiosus.
- Biochemical assays to determine nuclease activity and ATP dependence.
Main Results:
- A large, heat-stable complex of Mre11 and Rad50 was isolated.
- The complex exhibits single-strand endonuclease activity.
- ATP-dependent double-strand-specific exonuclease activity was demonstrated.
Conclusions:
- Confirms the identification and function of Pyrococcus furiosus Mre11 and Rad50 homologues.
- Demonstrates that functional Mre11-Rad50 complexes with similar biochemical properties exist in all life kingdoms.