Volume 127, Issue 6, Pages 1678-1684 (December 2004) The mismatch repair complex hMutSα recognizes 5-fluorouracil-modified DNA: Implications for chemosensitivity and resistance  Akihiro Tajima, Martin T. Hess, Betty L. Cabrera, Richard D. Kolodner, John M. Carethers  Gastroenterology  Volume 127, Issue 6, Pages 1678-1684 (December 2004) DOI: 10.1053/j.gastro.2004.10.001 Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 1 (A) Purity of baculovirus-synthesized hMutSα on coomassie staining of electrophoresed protein extracts. Extracts from MMR-proficient SW480 colon cancer cells (left lane), crude baculovirus-synthesized proteins (middle lane), and FPLC-purified baculovirus-synthesized proteins (right lane) were electrophoresed on a 7.5% PAGE gel and stained with 0.1% coomassie blue. Note the retention of 160-kilodalton and 105-kilodalton protein bands in FPLC-purified extracts, which, by Western blotting, corresponded to hMSH6 and hMSH2 proteins. (B) Immunoprecipitation of hMutSα with hMSH2 and hMSH6 antibodies. Crude baculovirus-produced proteins were precipitated with either anti-hMSH2 or anti-hMSH6 and blotted with both antibodies. Antibody to hMSH6 recognized immunoprecipates from anti-hMSH6 or anti-hMSH2 (top panel). Likewise, antibody to hMSH2 recognized immunoprecipitates from anti-hMSH2 or anti-hMSH6 (bottom panel). Gastroenterology 2004 127, 1678-1684DOI: (10.1053/j.gastro.2004.10.001) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 2 Electromobility gel shift assays utilizing purified hMutSα and complementary, C/T-mismatched, or 5-FU-modified oligonucleotides. Radiolabled ologonucleotides were incubated without or with purified hMutSα in the presence or absence of 4 mmol/L ATP. In A, complementary “matched” DNA or oligonucleotides with a C/T mispair was used. In B, the 5-FU-modified oligonucleotide was used. Note the acquisition of an oligo-hMutSα band with the C/T mismatch (A, lane 5) or 5-FU-modified DNA (B, lane 2) when incubated with purified hMutSα, and the dissolution of the complex with the addition of ATP (A, lane 6; B, lane 3). Gastroenterology 2004 127, 1678-1684DOI: (10.1053/j.gastro.2004.10.001) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 3 Biosensor analysis (surface plasmon resonance) of the association between hMutSα and complementary (curves E and F), C/T-mismatched (curves C and D), or 5-FU-modified oligonucleotides (curves A and B). Curves G and H are controls without DNA substrate. Biotinylated double-stranded oligonucleotides were bound to the streptavidin-coated sensor, and purified hMutSα was added in running buffer with (curves B, D, F, H) or without ATP (curves A, C, E, G). Note the relatively higher binding of hMutSα to the C/T mispair compared with complementary DNA and even higher binding to 5-FU-modified DNA. With ATP present in the running buffer, binding is diminished between hMutSα and the DNA substrates. Gastroenterology 2004 127, 1678-1684DOI: (10.1053/j.gastro.2004.10.001) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 4 Biosensor analysis (surface plasmon resonance) of the association (left curves) and ATP-induced dissociation (right curves) between hMutSα and complementary (curve C), C/T mismatched (curve B), or 5-FU-modified oligonucleotides (curve A). A dissociation buffer containing 4 mmol/L ATP was added (arrow) after 5 minutes of association for dissolution experiments. Note the initial rapid dissociation upon the addition of ATP, followed by a slower continue dissolution phase lasting more than 5 minutes to reach steady state in the 5-FU-modified and C/T mispair DNA substrates. Steady state was reached for complementary DNA within 2 minutes after ATP was added. Controls (curves D and E) indicate the specificity of ATP’s effect when hMutSα is bound to a DNA substrate compared with when the DNA substrate is absent. Gastroenterology 2004 127, 1678-1684DOI: (10.1053/j.gastro.2004.10.001) Copyright © 2004 American Gastroenterological Association Terms and Conditions