High affinity cooperative DNA binding by the yeast Mlh1-Pms1 heterodimer.
We demonstrate here that the Saccharomyces cerevisiae Mlh1-Pms1 heterodimer required for DNA mismatch repair and other cellular processes is a DNA binding protein. Binding was evaluated using a variety of single and double-stranded DNA molecules. Mlh1-Pms1 bound short substrates with low affinity and showed a slight preference for single-stranded DNA. ... In contrast, Mlh1-Pms1 exhibited a much higher affinity for long DNA molecules, suggesting that binding is cooperative. High affinity binding required a duplex DNA length greater than 241 base-pairs. The rate of association with DNA was rapid and dissociation of protein-DNA complexes following extensive dilution was very slow. However, in competition experiments, we observed a rapid active transfer of Mlh1-Pms1 from labeled to unlabeled DNA. Binding was non-sequence specific and highly sensitive to salt type and concentration, suggesting that Mlh1-Pms1 primarily interacts with the DNA backbone via ionic contacts. Cooperative binding was observed visually by atomic force microscopy as long, continuous tracts of Mlh1-Pms1 protein bound to duplex DNA. These images also showed that Mlh1-Pms1 simultaneously interacts with two different regions of duplex DNA. Taken together, the atomic force microscope images and DNA binding assays provide strong evidence that Mlh1-Pms1 binds duplex DNA with positive cooperativity and that there is more than one DNA binding site on the heterodimer. These DNA binding properties of Mlh1-Pms1 may be relevant to its participation in DNA mismatch repair, recombination and cellular responses to DNA damage.
Mesh Terms:
Adaptor Proteins, Signal Transducing, Allosteric Regulation, Base Pair Mismatch, Base Pairing, Binding, Competitive, Carrier Proteins, DNA, DNA Repair, DNA, Single-Stranded, DNA-Binding Proteins, Dimerization, Fungal Proteins, Microscopy, Atomic Force, Protein Binding, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Salts, Substrate Specificity, Thermodynamics
Adaptor Proteins, Signal Transducing, Allosteric Regulation, Base Pair Mismatch, Base Pairing, Binding, Competitive, Carrier Proteins, DNA, DNA Repair, DNA, Single-Stranded, DNA-Binding Proteins, Dimerization, Fungal Proteins, Microscopy, Atomic Force, Protein Binding, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Salts, Substrate Specificity, Thermodynamics
J. Mol. Biol.
Date: Sep. 28, 2001
PubMed ID: 11575920
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