Related Experiment Videos
Differential and simultaneous adenosine di- and triphosphate binding by MutS
Keith P Bjornson1, Paul Modrich
1Department of Biochemistry and Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710, USA.
The Journal of Biological Chemistry
|March 8, 2003
Summary
MutS protein has one high-affinity ADP site and one high-affinity nonhydrolyzable ATP analogue site per dimer. This suggests ATP.MutS.ADP is the active form during DNA mismatch repair.
Area of Science:
- Molecular biology
- Biochemistry
- DNA repair mechanisms
Background:
- The precise roles of ATP binding and hydrolysis in MutS function during DNA mismatch repair remain unclear.
- Understanding these roles is crucial for elucidating DNA repair pathways.
Purpose of the Study:
- To investigate the binding characteristics of adenosine diphosphate (ADP) and adenosine triphosphate (ATP) analogues to the MutS protein.
- To determine the affinities and stoichiometry of nucleotide binding sites within MutS.
Main Methods:
- Nitrocellulose filter binding assays were employed to quantify nucleotide binding.
- Equilibrium fluorescence anisotropy measurements were used to assess binding affinities.
- Fluorescence energy transfer assays provided insights into simultaneous binding events.
Main Results:
- MutS exhibits one high-affinity binding site for ADP and one for nonhydrolyzable ATP analogues per dimer.
- Low concentrations of 5'-adenylylimidodiphosphate (AMPPNP) facilitate ADP binding, requiring excess AMPPNP for displacement.
- ADP and nonhydrolyzable ATP analogues can bind concurrently to adjacent MutS subunits.
Conclusions:
- The MutS protein likely exists as an ATP.MutS.ADP ternary complex in solution.
- This ternary complex is potentially the functional form of MutS involved in DNA interactions in vivo.
- These findings clarify the nucleotide-binding properties of MutS in DNA mismatch repair.