The Phe-X-Glu DNA binding motif of MutS. The role of hydrogen bonding in mismatch recognition.
The crystal structures of MutS protein from Thermus aquaticus and Escherichia coli in a complex with a mismatch-containing DNA duplex reveal that the Glu residue in a conserved Phe-X-Glu motif participates in a hydrogen-bonded contact with either an unpaired thymidine or the thymidine of a G-T base-base mismatch. Here, the role of hydrogen bonding in mismatch recognition by MutS is assessed. The relative affinities of MutS for DNA duplexes containing nonpolar shape mimics of A and T, 4-methylbenzimidazole (Z), and difluorotoluene (F), respectively, that lack hydrogen bonding donors and acceptors, are determined in gel mobility shift assays. The results provide support for an induced fit mode of mismatch binding in which duplexes destabilized by mismatches are preferred substrates for kinking by MutS. Hydrogen bonding between the O epsilon 2 group of Glu and the mismatched base contributes only marginally to mismatch recognition and is significantly less important than the aromatic ring stack with the conserved Phe residue. A MutS protein in which Ala is substituted for Glu(38) is shown to be defective for mismatch repair in vivo. DNA binding studies reveal a novel role for the conserved Glu residue in the establishment of mismatch discrimination by MutS.
Schofield
M J
MJ
Genetics & Biochemistry Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
Brownewell
F E
FE
Nayak
S
S
Du
C
C
Kool
E T
ET
Hsieh
P
P
D000251:Adenosine Triphosphatases; D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D020137:Base Pair Mismatch; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D017931:DNA Primers; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism*; D006860:Hydrogen Bonding; D051719:MutS DNA Mismatch-Binding Protein; D010649:Phenylalanine / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D018698:Glutamic Acid; D010649:Phenylalanine; D004247:DNA; D000251:Adenosine Triphosphatases; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli