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Updated: Jul 15, 2026
Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein
Published on: March 31, 2010
Modulation of MutS ATP-dependent functional activities by DNA containing a cisplatin compound lesion (base damage and
Yuliya Sedletska1, Laurence Fourrier, Jean-Marc Malinge
1Centre de Biophysique Moléculaire, CNRS, Rue Charles Sadron, 45071 Orléans Cedex 02, France.
Abstract:
DNA damage-dependent signaling by the DNA mismatch repair (MMR) system is thought to mediate cytotoxicity of the anti-tumor drug cisplatin through molecular mechanisms that could differ from those required for normal mismatch repair. The present study investigated whether ATP-dependent biochemical properties of Escherichia coli MutS protein differ when the protein interacts with a DNA oligonucleotide containing a GT mismatch versus a unique site specifically placed cisplatin compound lesion, a cisplatin 1,2-d(GpG) intrastrand cross-link with a mispaired thymine opposite the 3' platinated guanine. MutS exhibited substantial affinity for this compound lesion in hydrolytic and in non-hydrolytic conditions of ATP, contrasting with the normal nucleotide inhibition effect of mispair binding. The cisplatin compound lesion was also shown to stimulate poorly MutS ATPase activity to approach the hydrolysis rate induced by nonspecific DNA. Moreover, MutS undergoes distinct conformation changes in the presence of the compound lesion and ATP under hydrolytic conditions as shown by limited proteolysis. In the absence of MutS, the cisplatin compound lesion was shown to induce a 39 degrees rigid bending of the DNA double helix contrasting with an unbent state for DNA containing a GT mispair. Furthermore, an unbent DNA substrate containing a monofunctional adduct mimicking a cisplatin residue failed to form a persistent nucleoprotein complex with MutS in the presence of adenine nucleotide. We propose that DNA bending could play a role in MutS biochemical modulations induced by a compound lesion and that cisplatin DNA damage signaling by the MMR system could be modulated in a direct mode.
Insights
The DNA mismatch repair (MMR) system
Area of Science:
- Molecular biology
- Biochemistry
- Genetics
Background:
- The DNA mismatch repair (MMR) system is crucial for DNA repair and mediates the cytotoxic effects of anti-tumor drugs like cisplatin.
- The precise molecular mechanisms by which MMR signals cisplatin-induced DNA damage are not fully understood and may differ from standard mismatch repair.
- Escherichia coli MutS protein is a key component of the MMR system involved in recognizing DNA lesions.
Purpose of the Study:
- To investigate the differential ATP-dependent biochemical properties of E. coli MutS when binding to a GT mismatch versus a specific cisplatin-induced DNA lesion.
- To explore the conformational changes and DNA bending induced by the cisplatin lesion in the presence of MutS and ATP.
- To elucidate the role of DNA bending in MutS modulation by cisplatin adducts and its implications for MMR-mediated signaling.
Main Methods:
- Biochemical assays were used to assess MutS protein affinity and ATPase activity in the presence of ATP under hydrolytic and non-hydrolytic conditions.
- Limited proteolysis was employed to detect conformational changes in MutS upon binding to the cisplatin lesion and ATP.
- DNA structural analysis techniques were used to measure DNA bending induced by the cisplatin lesion and MutS binding.
Main Results:
- MutS exhibited significant affinity for the cisplatin lesion, even under non-hydrolytic ATP conditions, unlike its interaction with GT mismatches.
- The cisplatin lesion poorly stimulated MutS ATPase activity, approaching rates seen with non-specific DNA.
- MutS underwent distinct conformational changes when interacting with the cisplatin lesion and ATP.
- The cisplatin lesion induced a significant 39-degree bend in the DNA double helix, while a GT mismatch did not.
- A monofunctional adduct mimicking a cisplatin residue failed to form a stable complex with MutS.
Conclusions:
- DNA bending induced by cisplatin adducts plays a role in modulating MutS biochemical activity.
- The MMR system's signaling of cisplatin DNA damage may involve direct interactions with specific lesion-induced DNA structures.
- These findings suggest a distinct mechanism for MMR-mediated cytotoxicity of cisplatin compared to standard mismatch repair.
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