Mismatch Recognition and DNA-dependent Stimulation of the ATPase Activity of hMutSα Is Abolished by a Single Mutation in the hMSH6 Subunit

Abstract

The most abundant mismatch binding factor in human cells, hMutS␣, is a heterodimer of hMSH2 and hMSH6, two homologues of the bacterial MutS protein.The C-terminal portions of all MutS homologues contain an ATP binding motif and are highly conserved throughout evolution. Although the N termini are generally divergent, they too contain short conserved sequence elements. A phenylalanine 3 alanine substitution within one such motif, GXFY(X) 5 DA, has been shown to abolish the mismatch binding activity of the MutS protein of Thermus aquaticus (Malkov, V. A., Biswas, I., CameriniOtero, R. D., and Hsieh, P. (1997) J. Biol. Chem. 272, 23811-23817). We introduced an identical mutation into one or both subunits of hMutS␣. The Phe 3 Ala substitution in hMSH2 had no effect on the biological activity of the heterodimer. In contrast, the in vitro mismatch binding and mismatch repair functions of hMutS␣ were severely attenuated when the hMSH6 subunit was mutated. Moreover, this variant heterodimer also displayed a general DNA binding defect. Correspondingly, its ATPase activity could not be stimulated by either heteroduplex or homoduplex DNA. Thus the N-terminal portion of hMSH6 appears to impart on hMutS␣ not only the specificity for recognition and binding of mismatched substrates but also the ability to bind to homoduplex DNA. Postreplicative mismatch repair (MMR)1 plays a key role in the maintenance of genomic integrity by eliminating base-base mismatches and insertion-deletion loops (IDLs) that arise in DNA during replication or recombination (1, 2). The repair process consists of four principal steps: (i) mismatch recognition, (ii) repairosome assembly, (iii) degradation of the newly synthesized strand from the strand discrimination signal to and past the mismatch, and (iv) resynthesis of the excised DNA tract (1). The mismatch recognition step is of particular interest; numerous in vitro and in vivo experiments, carried out over the past decade, have shown that the MMR system is capable of addressing a large number of substrates ranging from purine-pyrimidine, purine-purine, and pyrimidine-pyrimidine mismatches to IDLs of one to more than 10 extrahelical nucleotides (3-9). How are these various substrates recognized? In Escherichia coli the role of the mismatch recognition factor is played by a homodimer of the MutS protein, which can initiate the repair of base-base mismatches (5, 10) and IDLs up to four nucleotides (7). In human cells, this function is fulfilled by one of two heterodimers consisting of MutS homologues (MSH): hMutS␣, a heterodimer of hMSH2 and hMSH6 (11-13), which acts in the correction of base-base mismatches and small IDLs (8,12,14), and hMutS␤, a heterodimer of hMSH2 and hMSH3, which addresses principally IDLs (8, 14, 15). The situation in Saccharomyces cerevisiae is similar (16 -19).The highly conserved C termini of MutS homologue proteins contain the ATP binding sites as well as helix-turn-helix motifs (20 -22). As nucleotide binding alters the conformation of the DNA-bound factors, such that they...

Specialized mismatch repair function of Glu339 in the Phe-X-Glu motif of yeast Msh6

“…Only Msh6 has the Phe-X-Glu motif containing the conserved phenylalanine and glutamate residues that make base specific contacts with mismatched DNA in bacterial MutS, implying that Msh6 is the subunit responsible for contacting mismatched bases in the Msh2-Msh6 heterodimer. This implication is strongly supported by studies demonstrating that substituting other amino acids for the conserved phenylalanine in Msh6 results in reduced Msh2-Msh6 binding to mismatched DNA and strongly elevated mutation rates in cells [8,9,16]. In other studies, replacing the conserved glutamate in Saccharomyces cerevisiae Msh6 (Glu339) with alanine resulted in an increase in the rate of single base deletions in a run of 14 A-T base pairs [16,17].…”

“…Only a phenylalanine and a glutamate in one monomer of MutS directly contact the mismatched base, and both are part of a Phe-X-Glu motif that is conserved in many bacterial and eukaryotic MutS proteins [4][5][6][7]. The phenylalanine stacks with a mismatched or unpaired base and is essential for mismatch binding and for MMR function in E. coli and in eukaryotes [8][9][10][11]. The side chain of the glutamate also interacts with the mismatched base, including forming a hydrogen bond to purines or pyrimidines in base-base mismatches as well as in single-base insertion-deletion mismatches [4][5][6][7].…”

DNA mismatch repair: Molecular mechanism, cancer, and ageing

“…In the crystal structures, the DNA is sharply kinked about 60° towards a narrowed major groove at the mismatch. A conserved Phe-X-Glu motif at the N-terminus of bacterial MutS, originally identified in cross-linking studies, and eukaryotic MSH6 constitutes the mismatch binding motif with the Phe residue involved in an aromatic ring stack with the mismatched base displaced 2-3 Å into a widened minor groove (Dufner et al, 2000;Malkov et al, 1997). Mutation of the Phe abrogates mismatch binding in vitro and confers loss of MMR in vivo (Bowers et al, 1999;Das Gupta and Kolodner, 2000;Drotschmann et al, 2001;Yamamoto et al, 2000).…”

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

“…For instance, subunits of the E. coli MutS homodimer also exhibit differences in their interactions with nucleotides and with mismatched DNA [20,21]. The eukaryotic Msh2-Msh6 heterodimer is no different, as the subunits bind nucleotides with differing affinities [19,26], only one subunit catalyzes rapid ATP hydrolysis (Saccharomyces cerevisiae Msh2-Msh6: 2-3 s −1 at 20 °C) [19], and only Msh6 contains the conserved phenylalanine residue that can make specific contact with the mismatch in DNA [27,28]. As in the case of T.…”