Rapid communicationsThe mismatch repair complex hMutSα recognizes 5-fluorouracil-modified DNA: Implications for chemosensitivity and resistance
Section snippets
Synthesis of 5-FU-containing oligonucleotides
We synthesized 38mers in an oligonucleotide synthesizer (ABI, Foster City, CA) at the UCSD Cancer Center Oligonucleotide Synthesis Core. For a negative control (ie, perfect complement), we made 5′-TTTCTGACTTGGATACCATCTATCTATCTATAAAATAT-3′ and, for a positive control, we made 5′-TTTCTGACTTGTATACCATCTATCTATCTATAAAATAT-3′, which differs from the perfect complement at the twelfth nucleotide (G to T substitution). To synthesize the 5-FU-containing DNA, we utilized the phosphoramadite form of
Purification of recombinant hMutSα heterodimers
By coinfection of Sf9 cells with both the hMSH2 and hMSH6 baculovirus constructs, we were able to purify both proteins of the hMutSα complex as a heterodimer. This approach is crucial with respect to the stability of the proteins because individual MMR proteins tend to degrade rapidly.43 After sequential purification over 2 FPLC columns, hMSH2 and hMSH6 are visualized by coomassie staining as 160-kilodalton and 105-kilodalton bands (Figure 1A, lane 3). The level of purification is shown by
Discussion
Here, we demonstrate that hMutSα, a key MMR protein complex that recognizes base-base mispairs and small insertion-deletion loops for repair, recognizes and binds 5-FU-modifed DNA. The binding of 5-FU was reversed on addition of ATP, suggesting a functional interaction between hMutSα and 5-FU adducts. The potential recognition of 5-FU by the MMR system was investigated because of observed in vitro differences in cell survival with 5-FU treatment39, 40 and the publication of 2 retrospective
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Supported by the California Department of Health Services Cancer Research Program (grant 99-86873 to J.M.C.) and the United States Public Health Service (R01-CA90231 to J.M.C.).