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 containing mispaired or unpaired bases. To identify regions of MutS protein in close proximity to the heteroduplex DNA, we have utilized the photoactivated cross-linking moiety 5-iododeoxyuridine (5-IdUrd). Nucleoprotein complexes of Thermus aquaticus MutS protein bound to monosubstituted 5-IdUrd-containing heteroduplex DNAs were cross-linked with long-wavelength ultraviolet light. Positioning of the 5-IdUrd moiety at one of three positions within the DNA bulge, two nucleotides upstream or three nucleotides downstream of the unpaired base, resulted in an identical subset of cross-linked peptides as determined by proteolytic fingerprinting. 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 ability to dimerize, its thermostability, or its ATPase activity. These results implicate the region in the vicinity of Phe-39 as being crucial for heteroduplex DNA binding by Taq MutS protein.
Malkov
V A
VA
Genetics and Biochemistry Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-1810, USA.
Biswas
I
I
Camerini-Otero
R D
RD
Hsieh
P
P
D000251:Adenosine Triphosphatases; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D051719:MutS DNA Mismatch-Binding Protein; D016297:Mutagenesis, Site-Directed; D009692:Nucleic Acid Heteroduplexes; D010777:Photochemistry; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D013824:Thermus / Q000737:chemistry*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D009692:Nucleic Acid Heteroduplexes; D004247:DNA; D000251:Adenosine Triphosphatases; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli