Gastroenterology

Gastroenterology

Volume 127, Issue 6, December 2004, Pages 1678-1684
Gastroenterology

Rapid communications
The mismatch repair complex hMutSα recognizes 5-fluorouracil-modified DNA: Implications for chemosensitivity and resistance

A portion of this work was presented in abstract form at the annual meeting of the American Gastroenterological Association in Orlando, Florida, May 19, 2003.
https://doi.org/10.1053/j.gastro.2004.10.001Get rights and content

Background & Aims:: Recent evidence suggests that patients with advanced microsatellite unstable (MSI) colorectal cancers lack a survival benefit with 5-fluorouracil (5-FU)-based chemotherapy. Additionally, tumor cells with MSI (caused by defective DNA mismatch repair) are more resistant to 5-FU in culture compared with microsatellite stable cells, despite similar amounts of 5-FU incorporation into the cell’s DNA. We examined whether the component of the DNA mismatch repair (MMR) system that normally recognizes single base pair mismatches could specifically recognize 5-FU incorporated into DNA as a potential mechanism for chemosensitivity. Methods: We synthesized oligonucleotides with and without incorporated 5-FU and created oligonucleotides with a single base pair mismatch (as a positive control) to perform electromobility gel shift assays (EMSA) with a purified, baculovirus-synthesized hMutSα MMR complex. We also utilized surface plasmon resonance to measure relative binding differences between the oligonucleotides and hMutSα in real time. Results: Using EMSA, we demonstrate that hMutSα recognizes and binds 5-FU-modified DNA. The reaction is specific as added ATP dissociates the hMutSα complex from the 5-FU-modified strand. Using surface plasmon resonance, we demonstrate greater binding between hMutSα and 5-FU-modified DNA compared with complementary DNA or DNA containing a C/T mismatch. Conclusions: The MMR complex hMutSα specifically recognizes and binds to 5-FU-modified DNA. Because MMR components are required for the induction of apoptosis by many DNA-damaging agents, the chemosensitivity of 5-FU for patients with advanced colorectal cancer may be in part due to recognition of 5-FU incorporated into tumor DNA by the MMR proteins.

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.).

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