Figure 1.

Representative single-molecule FRET traces for MutS and MutS-E41A bound to GT, T-bulge, and CC mismatch DNA. (A) Models illustrating the interactions between Phe39 and Glu41 of Taq MutS and a GT mismatch or a T-bulge. The Taq MutS–T-bulge structure is derived from Protein Data Bank entry 1EWQ.¹¹ The Taq MutS–GT structure is a model derived from aligning the recognition motifs of E. coli MutS GT (Protein Data Bank entry 1E3M)¹⁰ with the Taq MutS–T-bulge structure (Protein Data Bank entry 1EWQ).¹¹ Molecular models were created using PyMOL Molecular Graphics System, version 1.3 (Schrödinger, LLC). (B) Cartoon illustrating the 50 bp DNA substrate used in single-molecule FRET studies. DNA was immobilized via a biotin and streptavidin surface functionalization. A TAMRA or Cy3 donor dye is located at the 3′ end, and a Cy5 acceptor dye is located 19 bases from the donor. (C and D) Example smFRET traces for MutS and MutS-E41A, respectively, in the presence of (i) GT, (ii) T-bulge, and (iii) CC. Acceptor or donor blinking causes excursions to zero FRET in some traces. Each tick mark on the x-axis represents 20 seconds.