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.
Hall
M C
MC
Laboratories of Molecular Genetics and Structural Biology, NIEHS, RTP, NC 27709, USA.
Wang
H
H
Erie
D A
DA
Kunkel
T A
TA
D048868:Adaptor Proteins, Signal Transducing; D000494:Allosteric Regulation; D020137:Base Pair Mismatch; D020029:Base Pairing; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D019281:Dimerization; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D018625:Microscopy, Atomic Force; D000070957:MutL Protein Homolog 1; D000070956:MutL Proteins; D011485:Protein Binding / Q000187:drug effects; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D012492:Salts / Q000494:pharmacology; D013379:Substrate Specificity; D013816:Thermodynamics
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C088859:MLH1 protein, S cerevisiae; C088858:PMS1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D012492:Salts; D004247:DNA; D000070957:MutL Protein Homolog 1; D000070956:MutL Proteins