Mismatch recognition and DNA-dependent stimulation of the ATPase activity of hMutS alpha is abolished by a single mutation in the hMSH6 subunit

The most abundant mismatch binding factor in human cells, hMutS alpha, is a heterodimer of hMSH2 and hMSH6, two homologues of the bacterial MutS prote in. The C-terminal portions of all MutS homologues contain an ATP binding m otif and are highly conserved throughout evolution. Although the N termini are generally divergent, they too contain short conserved sequence elements . A phenylalanine --> alanine substitution within one such motif, GXFY(X)(5 )DA, has been shown to abolish the mismatch binding activity of the MutS pr otein of Thermus aquaticus (Malkov, V. A, Biswas, I., Camerini-Otero, R, D. , and Hsieh, P, (1997) J. Biol. Chem. 272, 23811-23817); We introduced an i dentical mutation into one or both subunits of hMutS alpha The Phe --> Ala substitution in hMSH2 had no effect on the biological activity of the heter odimer. In contrast, the in vitro mismatch binding and mismatch repair func tions of hMutS alpha were severely attenuated when the hMSH6 subunit was mu tated. Moreover, this variant heterodimer also displayed a general DNA bind ing 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 alpha: not only the specificity for recognition and binding of mismatched substrates but also the ability to bind to homod uplex DNA.