Figure 1 The oligonucleotide sequence containing the T-bulge which was investigated by Natrajan, et al. The thymidine of the T-bulge is denoted in bold. 5’-AGC TGC CA G GCA CCA GTG TCA GCG TCC TAT-3’ 3’-TCG ACG GTTC CGT GGT CAC AGT CGC AGG ATA-5’

Figure 2 The key sequences of oligonucleotides used in the experiments proposed a)-c) The T-bulge oligonucleotides surrounded by adenine and thymidine d)-f) The T-bulge oligonucleotides surrounded by guanine and cytosine All sequences have the T-bulge denoted in bold type g)-h) The mismatch oligonucleotides, both sequences have the mismatch denoted in bold type B A E D C F 5’-ATA GGA CGC TGA CAC TGG TGC ATTG GCA GCT-3’ 3’-TAT CCT GCG ACT GTG ACC ACG T AC CGT CGA-5’ 5’-ATA GGA CGC TGA CAC TGG TGC GTCG GCA GCT-3’ 3’-TAT CCT GCG ACT GTG ACC ACG C GC CGT CGA-5’ 5’-ATA GGA CGC TGA CAC TGG TGC CTCG GCA GCT-3’ 3’-TAT CCT GCG ACT GTG ACC ACG G GC CGT CGA-5’ 5’-ATA GGA CGC TGA CAC TGG TGC GTGG GCA GCT-3’ 3’-TAT CCT GCG ACT GTG ACC ACG C CC CGT CGA-5’ 5’-ATA GGA CGC TGA CAC TGG TGC ATAG GCA GCT-3’ 3’-TAT CCT GCG ACT GTG ACC ACG T TC CGT CGA-5’ 5’-ATA GGA CGC TGA CAC TGG TGC TTTG GCA GCT-3’ 3’-TAT CCT GCG ACT GTG ACC ACG A AC CGT CGA-5’ G H 3’-TAT CCT GCG ACT GTG ACC ACG TAC CGT CGA-5’ 5’-ATA GGA CGC TGA CAC TGG TGC GTG GCA GCT-3’ 5’-ATA GGA CGC TGA CAC TGG TGC TTG GCA GCT-3’ 3’-TAT CCT GCG ACT GTG ACC ACG GAC CGT CGA-5’

Figure 3

Figure 4 B A C 5’-ATA GGA CGC TGA CAC TGG TGC XTXG GCA GCT-3’ 5’-TAT CCT GCG ACT GTG ACC ACG Y YC CGT CGA-3’ 5’-ATA GGA CGC TGA CAC TGG TGC XXG GCA GCT-3’ 5’-TAT CCT GCG ACT GTG ACC ACG YYC CGT CGA-3’ 5’-ATA GGA CGC TGA CAC TGG TGC XTXG GCA GCT-3’ 5’-TAT CCT GCG ACT GTG ACC ACG Y YC CGT CGA-3’ 5’-ATA GGA CGC TGA CAC TGG TGC XTXG GCA GCT-3’ 5’-TAT CCT GCG ACT GTG ACC ACG YAYC CGT CGA-3’ 5’-ATA GGA CGC TGA CAC TGG TGC ATAG GCA GCT-3’ 5’-TAT CCT GCG ACT GTG ACC ACG T TC CGT CGA-3’ 5’-ATA GGA CGC TGA CAC TGG TGC ATAG GCA GCT-3’ 5’-TAT CCT GCG ACT GTG ACC ACG T TC CGT CGA-3’ 5’-ATA GGA CGC TGA CAC TGG TGC ATAG GCA GCT-3’ 5’-TAT CCT GCG ACT GTG ACC ACG A TC CGT CGA-3’ Proposed pathways for each oligonucleotide investigated. X, Y = generic nucleotides; bidirectional arrow = hydrogen bond; T-bulge is denoted by bold type; new nucleotides are underlined A)Thymidine of T-bulge is excised B)Thymidine of T-bulge is paired with adenine C)Thymidine of T-bulge forms noncanonical base pair with thymidine which is “repaired” to thymidine by MutS complex

E D Figure 4, cont. Proposed pathways for each oligonucleotide investigated. X, Y = generic nucleotides; bidirectional arrow = hydrogen bond; T-bulge is denoted by bold type; new nucleotides are underlined D)Thymidine of T-bulge forms canonical base pair with adenine which is not recognized as a mismatch by the MutS complex; thus T-bulge is not repaired E) Thymidine of T-bulge is forms noncanonical base pair with cytosine which is “repaired” to adenine by MutS complex 5’-ATA GGA CGC TGA CAC TGG TGC TTTG GCA GCT-3’ 5’-TAT CCT GCG ACT GTG ACC ACG A AC CGT CGA-3’ 5’-ATA GGA CGC TGA CAC TGG TGC TTTG GCA GCT-3’ 5’-TAT CCT GCG ACT GTG ACC ACG A AC CGT CGA-3’ 5’-ATA GGA CGC TGA CAC TGG TGC TTTG GCA GCT-3’ 5’-TAT CCT GCG ACT GTG ACC ACG A AC CGT CGA-3’ 5’-ATA GGA CGC TGA CAC TGG TGC GTGG GCA GCT-3’ 5’-TAT CCT GCG ACT GTG ACC ACG C CC CGT CGA-3’ 5’-ATA GGA CGC TGA CAC TGG TGC GTGG GCA GCT-3’ 5’-TAT CCT GCG ACT GTG ACC ACG C CC CGT CGA-3’ 5’-ATA GGA CGC TGA CAC TGG TGC GTGG GCA GCT-3’ 5’-TAT CCT GCG ACT GTG ACC ACG A CC CGT CGA-3’

F Figure 4, cont. 5’-ATA GGA CGC TGA CAC TGG TGC CTCG GCA GCT-3’ 5’-TAT CCT GCG ACT GTG ACC ACG G GC CGT CGA-3’ 5’-ATA GGA CGC TGA CAC TGG TGC CTCG GCA GCT-3’ 5’-TAT CCT GCG ACT GTG ACC ACG G GC CGT CGA-3’ 5’-ATA GGA CGC TGA CAC TGG TGC CTCG GCA GCT-3’ 5’-TAT CCT GCG ACT GTG ACC ACG A GC CGT CGA-3’ Proposed pathways for each oligonucleotide investigated. X, Y = generic nucleotides; bidirectional arrow = hydrogen bond; T-bulge is denoted by bold type; new nucleotides are underlined D)Thymidine of T-bulge forms noncanonical base pair with guanine which is “repaired” to adenine by MutS complex

Figure 5 MutS complex Acid Quench Oligonucleotide Variable length reaction line for variable reaction time Ethanol precipitation and sequencing Diagram of the variable time reaction between MutS complex and each oligonucleotide of interest. Enzyme and oligonucleotide are injected into the reaction line from their respective containers. Acid quench is simultaneous injected to quench the reaction once it reaches the Quench Point (Q on the diagram). Q

Figure 6 C G Watson-Crick base pairs TA

Figure 7 B A A A T T T A A T T T Diagram of expected base pairings for T-bulge sequences A-C in complex with MutS. A) Sequence A; B) Sequence B; C) Sequence C

C Figure 7, cont. A A T T T Diagram of expected base pairings for T-bulge sequences A-C in complex with MutS. A) Sequence A; B) Sequence B; C) Sequence C

Figure 8 B A Diagram of expected contacts between MutS residues (blue, red, green, purple) and mismatched base pairs (black). Hydrogen bonds are denoted double headed arrows. A – Sequence G with MutS B – Sequence H with MutS

Table 1 Component1L Low (5mM) Imidazole Buffer 1L High (250mM) Imidazole Buffer 50mM dibasic Na 2 PO g dibasic Na 2 PO 4 500mM NaCl29.22g NaCl Imidazole2.5 mL 2M stock imidazole 0.01% Azide1 mL 10% Azide stock 10% glycerol100mL Water~800mL