The role of mismatched nucleotides in activating the hMSH2-hMSH6 molecular switch.
We have previously shown that hMSH2-hMSH6 contains an intrinsic ATPase which is activated by mismatch-provoked ADP-->ATP exchange that coordinately induces the formation of a sliding clamp capable of hydrolysis-independent diffusion along the DNA backbone (1,2). These studies suggested that mismatch repair could be propagated by a signaling event transduced via diffusion of ATP-bound hMSH2-hMSH6 molecular switches to the DNA repair machinery. The Molecular Switch model (Fishel, R. (1998) Genes Dev. 12, 2096-2101) is considerably different than the Hydrolysis-Driven Translocation model (Blackwell, L. J., Martik, D., Bjornson, K. P., Bjornson, E. S., and Modrich, P. (1998) J. Biol. Chem. 273, 32055-32062) and makes additional testable predictions beyond the demonstration of hydrolysis-independent diffusion (Gradia, S., Subramanian, D., Wilson, T., Acharya, S., Makhov, A., Griffith, J., and Fishel, R. (1999) Mol. Cell 3, 255-261): (i) individual mismatch-provoked ADP-->ATP exchange should be unique and rate-limiting, and (ii) the k(cat x DNA) for the DNA-stimulated ATPase activity should decrease with increasing chain length. Here we have examined hMSH2-hMSH6 affinity and ATPase stimulatory activity for several DNA substrates containing mispaired nucleotides as well as the chain length dependence of a defined mismatch under physiological conditions. We find that the results are most consistent with the predictions of the Molecular Switch model.
Gradia
S
S
Genetics and Molecular Biology Program, Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Acharya
S
S
Fishel
R
R
D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D020137:Base Pair Mismatch; D001667:Binding, Competitive; D004247:DNA / Q000494:pharmacology; D004260:DNA Repair / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019281:Dimerization; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D051718:MutS Homolog 2 Protein; D009838:Oligodeoxyribonucleotides / Q000378:metabolism / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D012492:Salts / Q000494:pharmacology
D004268:DNA-Binding Proteins; C094443:G-T mismatch-binding protein; D009838:Oligodeoxyribonucleotides; D011518:Proto-Oncogene Proteins; D012492:Salts; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004247:DNA; D000251:Adenosine Triphosphatases; C497172:MSH2 protein, human; D051718:MutS Homolog 2 Protein