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ATP-dependent assembly of a ternary complex consisting of a DNA mismatch and the yeast MSH2-MSH6 and MLH1-PMS1
Y Habraken1, P Sung, L Prakash
1Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1061, USA.
Abstract:
MSH2 and MSH6 proteins exist as a stable complex, as do the MLH1 and PMS1 proteins. To study the mismatch binding properties of the MSH2-MSH6 complex and to examine its functional interaction with the MLH1-PMS1 complex, these protein complexes were purified to near homogeneity from overproducing yeast strains. As has been reported previously, the purified MSH2-MSH6 complex binds DNA substrates containing a G/T mismatch and insertion/deletion mismatches, but the binding affinity for the latter decreases as the size of the extrahelical loop increases. Addition of ATP or the nonhydrolyzable ATPgammaS reduces binding of the MSH2-MSH6 complex to the DNA substrates markedly. Here, we show that MSH2-MSH6 forms a ternary complex with MLH1-PMS1 on a mismatch containing DNA substrate. The formation of this ternary complex requires ATP, which can be substituted by ATPgammaS, suggesting that ATP binding alone is sufficient for ternary complex formation. Thus, it appears that ATP binding by the MSH2-MSH6 complex induces a conformation that is conducive for the interaction with MLH1-PMS1 complex, leading to the formation of the ternary complex.
Insights
The MSH2-MSH6 and MLH1-PMS1 protein complexes interact to form a ternary complex on mismatched DNA. This interaction requires ATP binding, highlighting a key step in DNA mismatch repair.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- The MSH2-MSH6 and MLH1-PMS1 protein complexes are crucial for DNA mismatch repair.
- Understanding their interaction is key to elucidating DNA repair mechanisms.
Purpose of the Study:
- To investigate the functional interaction between the MSH2-MSH6 and MLH1-PMS1 complexes.
- To characterize the DNA mismatch binding properties of these complexes.
Main Methods:
- Purification of MSH2-MSH6 and MLH1-PMS1 protein complexes from yeast.
- Analysis of DNA substrate binding affinities.
- Investigation of ternary complex formation using ATP and ATPgammaS.
Main Results:
- MSH2-MSH6 binds G/T and insertion/deletion mismatches, with affinity decreasing for larger loops.
- ATP/ATPgammaS markedly reduces MSH2-MSH6 binding to DNA substrates.
- A ternary complex of MSH2-MSH6 and MLH1-PMS1 forms on mismatched DNA, requiring ATP binding.
Conclusions:
- ATP binding induces a conformational change in MSH2-MSH6, facilitating interaction with MLH1-PMS1.
- This interaction is essential for the formation of the ternary complex in DNA mismatch repair.