Assembly and Molecular Activities of the MutS Tetramer

Abstract

Analytical equilibrium ultracentrifugation indicates that Escherichia coli MutS exists as an equilibrating mixture of dimers and tetramers. The association constant for the dimer-to-tetramer transition is 2.1 ؋ 10 7 M ؊1 , indicating that the protein would consist of both dimers and tetramers at physiological concentrations. The carboxyl terminus of MutS is required for tetramer assembly because a previously described 53-amino acid carboxyl-terminal truncation (MutS800) forms a limiting species of a dimer (Obmolova, G., Ban, C., Hsieh, P., and Yang, W. MutS homologs participate in multiple genetic stabilization pathways by virtue of their ability to recognize a spectrum of DNA lesions. Recognition of base pair mismatches by MutS homologs has been implicated in the rectification of DNA biosynthetic errors and in the dissolution of recombination events involving diverged sequences (1-5). Recognition of damaged base pairs by MutS homologs is also involved in the transcription-coupled repair of DNA damage (2, 4) and has been implicated in the activation of checkpoint and apoptotic responses to certain types of DNA damage in mammalian cells (6). Members of the MutS family can thus be viewed as molecular sentinels that respond to subtle variations in DNA structure or dynamics and then communicate presence of that lesion to downstream activities (1, 7-9).Events downstream of MutS recognition have been delineated for the Escherichia coli pathway responsible for correction of DNA biosynthetic errors, and this reaction has been reconstituted in vitro using near homogeneous components. Although a number of mechanistic details remain to be established, basic roles of the individual activities have been defined. MutS recruits MutL to the heteroduplex in a reaction requiring ATP (10 -12). Assembly of this ternary complex is sufficient to activate the latent endonuclease activity of MutH, which incises the unmethylated strand at a hemimethylated d(GATC) strand signal (13). In addition, this complex activates the unwinding activity of DNA helicase II, which loads at the MutH incision with an orientation bias so that helix unwinding proceeds toward the mismatch (14, 15). The unwound portion of the incised strand is hydrolyzed by a single-strand exonuclease. If unwinding proceeds from a strand break located 5Ј to the mispair, the displaced single strand is degraded by the 5Ј-to-3Ј hydrolytic activity of ExoVII or RecJ exonuclease (16,17). When helicase unwinding initiates at a nick 3Ј to the mismatch, the unwound strand is degraded by the 3Ј-to-5Ј activity of ExoI, ExoVII,. Excision terminates at a number of sites centered about 50 base pairs beyond the mismatch, the ensuing gap is repaired by DNA polymerase III holoenzyme in the presence of single-strand binding protein, and DNA ligase restores covalent integrity to the repaired strand (18,20).In addition to its mismatch recognition activity, bacterial MutS harbors a weak ATPase (21) that is stimulated by DNA (22). Because the rate-limiting step for ATP hydrolytic turnover is se...

Hydrolytically Deficient MutS E694A Is Defective in the MutL-dependent Activation of MutH and in the Mismatch-dependent Assembly of the MutS · MutL · Heteroduplex Complex

“…The simplest interpretation of this value is that it corresponds to the intrinsic ATP hydrolytic rate constant for the mutant protein. This parameter is almost identical to the k cat for steady state ATP hydrolysis by MutS E694A at 30°C (see above), which is 0.08 min Ϫ1 when expressed per dimer, the minimum MutS functional unit (22,26,37,38). The similarity of these values indicates that hydrolytic chemistry is largely rate-limiting for turnover by MutS E694A.…”

“…ATPase, Mismatch Repair Assays, and Surface Plasmon Resonance Spectroscopy-Steady state MutS ATPase hydrolytic parameters and activation of the MutH d(GATC) endonuclease were determined as described previously (36,37), except that ATPase reactions were performed in 25 mM Tris-HCl, pH 7.6, 100 mM KCl, 5 mM MgCl 2 , 1 mM dithiothreitol, 0.1 mg/ml bovine serum albumin.…”

“…MutS E694A Is Defective in the MutL-dependent Activation of the MutH Endonuclease-Initiation of E. coli mismatch repair depends on the mismatch-, MutS-, and MutL-dependent activation of the MutH d(GATC) endonuclease (7). This activity has been scored in vitro using heteroduplexes in which the mismatch and d(GATC) site reside on the same DNA molecule (6,7,37), and by a trans activation assay in which the two DNA sites reside on separate synthetic oligonucleotide duplexes (23,34,40). The finding that a His-tagged version of MutS E694A supports MutH activation in the trans assay has led to the suggestion that ATP binding by MutS is sufficient for activation of downstream activities (23).…”

DNA bending and unbending by MutS govern mismatch recognition and specificity

“…Both E. coli and Taq MutS proteins have been shown to exist in an equilibrium of dimers and tetramers (21,37,38). This observation raises the question of whether the bent and unbent populations correlate with the different oligomeric states of MutS proteins.…”

DNA conformations in mismatch repair probed in solution by X-ray scattering from gold nanocrystals

“…coli MutS forms tetramers, with a reported K d for the dimer/ tetramer equilibrium ranging from 210 to 2,200 nM −1 (34,35), that could complicate the analysis of the P(D i,j ) distributions if mixed populations of dimers and tetramers were present. Use of the gold nanocrystals, however, allowed us to make measurements at protein concentrations as low as 50 nM, well below the K d .…”