Biochemical characterization of the interaction between the Saccharomyces cerevisiae MSH2-MSH6 complex and mispaired bases in DNA.

The interaction of the Saccharomyces cerevisiae MSH2-MSH6 complex with mispaired bases was analyzed using gel mobility shift assays and surface plasmon resonance methods. Under equilibrium binding conditions, MSH2-MSH6 bound to homoduplex DNA with a K(d) of 3.9 nM and bound oligonucleotide duplexes containing T:G, +1, +2, +4, and +10 insertion/deletion ...
loop (IDL) mispairs with K(d) values of 0.20, 0.25, 11, 3.2, and 0.55 nM, respectively. Competition binding experiments using 65 different substrates revealed a 10-fold range in mispair discrimination. In general, base-base mispairs and a +1 insertion/deletion mispair were recognized better than intermediate sized insertion/deletion mispairs of 2-8 bases. Larger IDL mispairs (>8 bases) were recognized almost as well as the +1 IDL mispair. Recognition of mispairs by MSH2-MSH6 was influenced by sequence context, with the 6-nucleotide region surrounding the mispair being primarily responsible for influencing mispair recognition. Effects of sequences as far away as 15 nucleotides were also observed. Differential effects of ATP on the stability of MSH2-MSH6-mispair complexes suggested that base-base mispairs and the smaller IDL mispairs were recognized by a different binding mode than larger IDL mispairs, consistent with genetic experiments indicating that MSH2-MSH6 functions primarily in the repair of base-base and small IDL mispairs.
Mesh Terms:
Adenosine Diphosphate, Adenosine Triphosphate, Base Pair Mismatch, Base Sequence, Biosensing Techniques, DNA Repair, DNA, Fungal, DNA-Binding Proteins, Fungal Proteins, Macromolecular Substances, Molecular Sequence Data, MutS Homolog 2 Protein, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins
J. Biol. Chem.
Date: Sep. 17, 1999
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