DNA-dependent Activation of the hMutSα ATPase

Abstract

ATP hydrolysis by MutS homologs is required for function of these proteins in mismatch repair. However, the function of ATP hydrolysis in the repair reaction is controversial. In this paper we describe a steady-state kinetic analysis of the DNA-activated ATPase of human MutS␣. Comparison of salt concentration effects on mismatch repair and mismatch-provoked excision in HeLa nuclear extracts with salt effects on the DNA-activated ATPase suggests that ATP hydrolysis by MutS␣ is involved in the rate determining step in the repair pathway. While the ATPase is activated by homoduplex and heteroduplex DNA, the half-maximal concentration for activation by heteroduplex DNA is significantly lower under physiological salt concentrations. Furthermore, at optimal salt concentration, heteroduplex DNA increases the k cat for ATP hydrolysis to a greater extent than does homoduplex DNA. We also demonstrate that the degree of ATPase activation is dependent on DNA chain length, with the k cat for hydrolysis increasing significantly with chain length of the DNA cofactor. These results are discussed in terms of the translocation

The Role of Mismatched Nucleotides in Activating the hMSH2-hMSH6 Molecular Switch

“…Homology between the MutS homologs is largely based upon a highly conserved Walker-A/B adenosine nucleotide and magnesium-binding domain (3,9). Although aspects of ATP binding/hydrolysis by the bacterial and yeast MutS homologs have been examined (15)(16)(17), more comprehensive studies of the human hMSH2-hMSH6 heterodimer have demonstrated coupled ATP and DNA binding properties as well as an intrinsic ATPase activity that is stimulated by mispaired DNA (1,2,5,18,19). A defining observation is that binding to mismatched DNA by MutS and hMSH2-hMSH6 is abolished in the presence of ATP (1,10,20).…”

“…An argument against the Molecular Switch model was recently forwarded by Blackwell et al (5,19) and is based on the following observations: (i) a similar dissociation constant (k d ) between hMSH2-hMSH6 and mismatched DNA in the presence or absence of ADP; (ii) plasmon resonance spectroscopy demonstrating ADP-induced release of hMSH2-hMSH6, which was prebound to mismatched DNA in the absence of nucleotide (albeit Ͼ10-fold slower than ATP-induced release); (iii) an ATPase "salt profile" that appeared similar to MMR and mismatch-provoked excision reactions in vitro, and 4.) a "significant" DNA length-dependent increase in the k cat⅐DNA for the ATPase.…”

“…An argument against the Molecular Switch model was recently forwarded by Blackwell et al (5,19) and is based on the following observations: (i) a similar dissociation constant (k d )…”

“…a "significant" DNA length-dependent increase in the k cat⅐DNA for the ATPase. It was concluded that hMSH2-hMSH6 movement along the DNA backbone occurred by a modified HydrolysisDriven Translocation model (5,19).…”

“…This conclusion is grounded on the biochemical properties of the hMSH2-hMSH6 ATPase and the observation that ADP and ATP have opposing effects on mispair binding. These studies have further demonstrated that: (i) the rate-limiting step for the intrinsic ATPase is mismatched nucleotide provoked ADP3 ATP exchange; (ii) hMSH2-hMSH6 undergoes a conformational transition associated with ADP3 ATP exchange similar to that demonstrated for G protein signaling molecules; (iii) the adenosine nucleotide conformational transition of hMSH2-hMSH6 results in the formation of an ATP-bound hydrolysis-independent sliding clamp (preincubation of hMSH2-hMSH6 with ATP␥S, results in a conformation that is topologically refractory to mispair binding); and (iv) hydrolysis of ATP only occurs when hMSH2-hMSH6 dissociates or is dissociated from the DNA (thus recycling the mispair binding switch).An argument against the Molecular Switch model was recently forwarded by Blackwell et al (5,19) and is based on the following observations: (i) a similar dissociation constant (k d ) …”