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'Saccharomyces cerevisiae MSH2/6 complex interacts with Holliday junctions and facilitates their cleavage by phage
G T Marsischky1, S Lee, J Griffith
1Charles A. Dana Division of Human Cancer Genetics, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.
Abstract:
Genetic and biochemical studies have indicated that mismatch repair proteins can interact with recombination intermediates. In this study, gel shift assays and electron microscopic analysis were used to show that the Saccharomyces cerevisiae MSH2/6 complex binds to Holliday junctions and has an affinity and specificity for them that is at least as high as it has as for mispaired bases. Under equilibrium binding conditions, the MSH2/6 complex had a Kd of binding to Holliday junctions of 0.5 nM. The MSH2/6 complex enhanced the cleavage of Holliday junctions by T4 endonuclease VII and T7 endonuclease I. This is consistent with the view that the MSH2/6 complex can function in both mismatch repair and the resolution of recombination intermediates as predicted by genetic studies.
Insights
The Saccharomyces cerevisiae MSH2/6 complex binds to Holliday junctions with high affinity, similar to its binding of mispaired bases. This suggests the MSH2/6 complex plays a role in both DNA mismatch repair and resolving recombination intermediates.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Mismatch repair proteins are known to interact with recombination intermediates.
- The Saccharomyces cerevisiae MSH2/6 complex is a key player in DNA mismatch repair.
Purpose of the Study:
- To investigate the interaction of the MSH2/6 complex with Holliday junctions.
- To determine the binding affinity and specificity of MSH2/6 for Holliday junctions.
- To assess the functional consequences of MSH2/6 binding to Holliday junctions.
Main Methods:
- Gel shift assays were employed to detect binding.
- Electron microscopic analysis provided structural insights.
- Enzyme cleavage assays were used to assess functional impact.
Main Results:
- The MSH2/6 complex exhibits high affinity (Kd = 0.5 nM) and specificity for Holliday junctions.
- Binding affinity for Holliday junctions is comparable to that for mispaired bases.
- The MSH2/6 complex enhanced the cleavage of Holliday junctions by T4 endonuclease VII and T7 endonuclease I.
Conclusions:
- The MSH2/6 complex directly binds to and influences the resolution of Holliday junctions.
- These findings support the dual role of the MSH2/6 complex in both mismatch repair and recombination intermediate resolution.
- The study provides biochemical evidence for genetically predicted functions of the MSH2/6 complex.