PHOTOCROSS-LINKING OF THE NH2-TERMINAL REGION OF TAQ MUTS PROTEIN TO THE MAJOR GROOVE OF A HETERODUPLEX DNA

The MutS DNA mismatch repair protein recognizes heteroduplex DNAs cont aining mispaired or unpaired bases, To identify regions of MutS protei n in close proximity to the heteroduplex DNA, we have utilized the pho toactivated cross-linking moiety 5-iododeoxyuridine (5-IdUrd), Nucleop rotein complexes of Thermus aquaticus MutS protein bound to monosubsti tuted 5-IdUrd-containing heteroduplex DNAs were cross-linked with long -wavelength ultraviolet light, Positioning of the 5-IdUrd moiety at on e of three positions within the DNA bulge, two nucleotides upstream or three nucleotides downstream of the unpaired base, resulted in an ide ntical subset of cross-linked peptides as determined by proteolytic fi ngerprinting, The tryptic peptide cross linked to an unpaired 5-IdUrd residue was determined by peptide sequencing to correspond to a highly conserved region spanning residues 25-49. Cross-linking to the bulge nucleotide occurred at Phe-39, indicating that this residue contacts, or is in close proximity to, the unpaired base of a heteroduplex DNA. Site-directed mutagenesis resulting in the substitution of Ala for Phe -39 reduced the affinity of the mutant protein for heteroduplex DNA by roughly 3 orders of magnitude, but had no apparent effect on its abil ity to dimerize, its thermostability, or its ATPase activity, These re sults implicate the region in the vicinity of Phe-39 as being crucial for heteroduplex DNA binding by Tag MutS protein.