Nucleotide-promoted Release of hMutSα from Heteroduplex DNA Is Consistent with an ATP-dependent Translocation Mechanism

Abstract

In addition to their mismatch recognition activities, bacterial and eukaryotic MutS activities have an associated ATPase activity that is required for function of the proteins in mismatch repair (1-5). Two distinct functions have been proposed for nucleotide binding and hydrolysis by MutS homologs, both of which are based on the effects of ATP on MutS-heteroduplex interaction. The presence of ATP greatly reduces the efficiency of specific complex formation between bacterial MutS or eukaryotic MutS␣ and heteroduplex DNA (5-10), and ATP challenge of preformed MutS⅐heteroduplex complexes has been shown to result in departure of the protein from the mismatch (11). Available information indicates that some of these effects are attributable to ATP binding. Thus, ATP␥S has been shown to promote departure of MutS from the mismatch in heteroduplex DNA (11), while ATP␥S or ATP (in the absence of a divalent cation) reduce the binding efficiency human MutS␣ (hMutS␣) to synthetic heteroduplexes (5, 10).Electron microscopy of complexes between bacterial MutS and large heteroduplexes prepared from natural DNAs has demonstrated that ATP-promoted release of MutS from a mismatch is associated with efficient conversion of protein⅐DNA complexes to ␣-shaped loop structures stabilized by MutS at the base (11). Loop formation requires a mismatch, loop size increases linearly with time, loop growth depends on continued ATP hydrolysis, and the mismatch usually ends up in the loop. These observations have been interpreted in terms of a mechanism in which ATP binding reduces affinity of the protein for a mispair and activates secondary DNA binding sites that are subsequently used for movement of the protein along the helix contour in a reaction dependent on nucleotide hydrolysis (11). MutS movement in this manner has been postulated to be important for the coupling of mismatch recognition to loading of the excision system at the strand break that directs repair (12, 13), a site that can be located hundreds of base pairs from this mismatch.The finding that ATP binding reduces the efficiency of specific complex formation between hMutS␣ and oligonucleotide heteroduplexes has led to proposal of a molecular switch model for action of MutS activities. Like a G-protein, hMutS␣ is postulated to exist in two states, an ADP-bound form that binds with near irreversible affinity to a mismatch and an ATPbound form that does not (10). In this proposal hMutS␣⅐ADP binds to a mispair and recruits downstream activities to this site. After assembly of the excision system, ATP binding results in dissociation of hMutS␣ from the heteroduplex so that repair may proceed (10).To further clarify the role(s) of ATP binding and hydrolysis in hMutS␣ action, we have evaluated the effects of ATP, ADP, and nonhydrolyzable ATP analogs on the lifetime of hMutS␣⅐DNA complexes and have examined the effect of DNA topology on ATP-promoted dissociation of hMutS␣ complexes with small heteroduplexes. We demonstrate that ADP is not required for mismatch recognition by hMutS␣, but...

Mismatch Recognition-Coupled Stabilization of Msh2-Msh6 in an ATP-Bound State at the Initiation of DNA Repair

“…The ATPase activity of bacterial (E. coli, Thermus aquaticus MutS) and eukaryotic (S. cerevisiae, human Msh2-Msh6) mismatch recognition proteins has been examined extensively under steady-state conditions, both in the absence and in the presence of DNA substrates (12,15,24,28). In general, matched and mismatched DNA substrates appear to stimulate the steadystate ATPase rate, although the extent of stimulation varies with the protein source and assay conditions (e.g., NaCl, temperature).…”

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.…”

Uncertain pathogenicity of MSH2 variants N127S and G322D challenges their classification

“…Neither variant showed defects in interaction with MSH6, mismatch binding activities, or MMR efficiency compared to wild-type MSH2. Furthermore, our preliminary experiments on kinetic properties of MSH2 variants indicate that the MSH2 N127S and G322D mutations do not interfere with the release of MutSa from the mismatch, which has been shown to occur upon ATP uptake 33 (data not shown). Overall, our functional results demonstrate that these variants do not compromise MMR at least when being the only MMR variation in a cell.…”