The Mismatch Repair Complex HMutS Alpha Recognizes 5-fluorouracil-modified DNA: Implications for Chemosensitivity and Resistance

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 hMutS alpha MMR complex. We also utilized surface plasmon resonance to measure relative binding differences between the oligonucleotides and hMutS alpha in real time.

Results: Using EMSA, we demonstrate that hMutS alpha recognizes and binds 5-FU-modified DNA. The reaction is specific as added ATP dissociates the hMutS alpha complex from the 5-FU-modified strand. Using surface plasmon resonance, we demonstrate greater binding between hMutS alpha and 5-FU-modified DNA compared with complementary DNA or DNA containing a C/T mismatch.

Conclusions: The MMR complex hMutS alpha 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.