hMSH2–hMSH6 Forms a Hydrolysis-Independent Sliding Clamp on Mismatched DNA

Abstract

Mismatch recognition by the human MutS homologs hMSH2-hMSH6 is regulated by adenosine nucleotide binding, supporting the hypothesis that it functions as a molecular switch. Here we show that ATP-induced release of hMSH2-hMSH6 from mismatched DNA is prevented if the ends are blocked or if the DNA is circular. We demonstrate that mismmatched DNA provokes ADP-->ATP exchange, resulting in a discernible conformational transition that converts hMSH2-hMSH6 into a sliding clamp capable of hydrolysis-independent diffusion along the DNA backbone. Our results support a model for bidirectional mismatch repair in which stochastic loading of multiple ATP-bound hMSH2-hMSH6 sliding clamps onto mismatch-containing DNA leads to activation of the repair machinery and/or other signaling effectors similar to G protein switches.

Contribution of Msh2 and Msh6 subunits to the asymmetric ATPase and DNA mismatch binding activities of Saccharomyces cerevisiae Msh2–Msh6 mismatch repair protein

“…MutS/Msh proteins bind mismatched DNA with high affinity and stability, but in the presence of ATP (and ATPγS), the interaction is altered and the protein dissociates from DNA if its ends are left unblocked [9,11,15]. MutS/Msh binding to DNA appears also to be sensitive to the concentration of NaCl in the reaction, as expected for protein-DNA interactions that involve sequence non-specific contacts.…”

“…ATP binding and hydrolysis appear to modulate the interactions between MutS/Msh and DNA as well as other proteins in the repair pathway; thus, understanding how MutS/Msh proteins utilize ATP is necessary for understanding how they function in DNA mismatch repair. Several model mechanisms have been proposed for MutS/Msh action upon mismatch recognition: (a) MutS/Msh proteins translocate on DNA, fuelled by ATP binding and hydrolysis, possibly to interact with other proteins on DNA and coordinate mismatch recognition with downstream events such as initiation of strand excision and DNA resynthesis [8][9][10]; (b) upon binding ATP MutS/Msh proteins form sliding clamps that diffuse freely on DNA, again, to contact downstream repair proteins and direct repair [11,12]; (c) MutS/Msh proteins utilize ATP binding and hydrolysis to modulate their interaction with DNA, while remaining at the mismatch to direct repair [13][14][15][16][17]. At present, experimental data are available in support of each of these very different model mechanisms, therefore the investigation into MutS/Msh DNA binding and ATPase activities continues.…”

Structure and function of ABC transporters: the ATP switch provides flexible control

“…Although, in other studies of P-gp, drug substrates have been reported not to alter the affinity for ATP [22,31], in these studies, the affinity for ATP was indirectly inferred from increased ATPase activity above a basal activity in the absence of added substrate; this basal activity itself reflects the anticipated futile cycle (see above), and so the data cannot be interpreted as intended. Finally, binding of substrate ('mismatch' DNA) to the DNA-binding domain (analogous to the TMDs of an ABC transporter) of the MutS mismatch repair enzyme stimulates the NBD to bind ATP [35,36].…”

“…For the cystic fibrosis transmembrane regulator, although initial studies suggested that ATP hydrolysis drives channel opening, a more detailed analysis has revealed that channel opening can be mediated by ATP binding to the NBDs in the absence of hydrolysis [49]. Similarly, for the DNA 'mismatch' repair ABC proteins, MutS and hMSH2/6, ATP binding in the absence of hydrolysis induces the key conformational change which enables the DNA repair complex to assemble [32,35,36].…”

“…This mechanism may not be common to all ABC NBDs, and, indeed, for MutS, ADP appears to remain bound to one NBD until substrate binding induces a conformational change in the NBDs, such that ADP is displaced by ATP [36]. For P-gp [22,56,57] and MutS [35], it has been shown that ADP release provides a rate-limiting step in the ATPase cycle.…”