It has been recently demonstrated that hMSH3, the recognition component of hMutS?, plays an important role in recognizing DNA damage, particularly interstrand crosslinks. Although the importance of this component of hMutS? has been recognized in furthering CRC progression, there is no data regarding hMutS? contributions in executing 5-FU toxicity, a key issue since all major therapy for CRC involves 5-FU. Our study demonstrates that 5-FU cytotoxicity depends on the hMutSa and hMutS? status of cells, with the most cytotoxicity observed when both hMutSa and hMutS? are present, intermediate 5-FU cytotoxicity when hMutSa is retained but hMutS? is deficient, and low 5-FU cytotoxicity when cells are hMutSa deficient but hMutS? proficient, that hMutS? recognizes 5-FU-containing DNA, and that the Abmole VE-822 binding affinity of hMutS? is lower than that of hMutSa, which directly correlates with 5-FU cytotoxicity. This is the first study demonstrating an additive role for 5-FU cytotoxicity for hMutSa and hMutS?. Our data indicate that 5-FU cytotoxicity is more severe in cells that retain both hMutSa and hMutS? than the cells that lack hMutSa and retain hMutS?, which support our prior data that hMutSa recognition of 5-FU incorporated into DNA plays an important role for 5-FU chemosensitivity. It is somewhat surprising that our EMSA results showed that a single base-pair of 5-FU incorporated into DNA is recognized by hMutS? in spite of its general role for recognition of I/D loops greater than 2 molecules, and not single mispairs. This could be due to something unique regarding 5-FU, such as its negative charge, or an atypical or unrecognized role for hMutS? in recognizing altered nucleotides. Takahashi et al. demonstrated that cells that lack hMSH3,a component of hMutS?, are more sensitive to cysplatin and oxaliplatin than Publications Using Abomle Cathepsin inhibitor 1 hMSH3-proficient cells. Interestingly, they demonstrated that the difference of chemosensitivity between hMSH3-deficient and hMSH3-proficient cells occurred independently of hMLH1 status. In addition, it has been shown that hMutS? recognizes ICLs induced by psoralen, and is not dependent on hMutSa or hMLH1. hMutS? was also reported to interact with nucleotide excision repair proteins, and homologous recombination repair level for ICLs is also dependent on hMutS? and not on hMutSa, which may suggest that hMutS? may cooperate with the NER or HR proteins for ICLs repair perhaps independently of traditional DNA MMR. It is possible that hMutS? recognition of 5-FU incorporated into DNA might trigger MMR-independent repair mechanisms on top of classical DNA MMR.