The mismatch repair complex hMutS alpha recognizes 5-fluorouracil-modified DNA: implications for chemosensitivity and resistance.
OBJECTIVE
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.
Tajima
Akihiro
A
Department of Medicine, University of California, La Jolla, California, USA.
Hess
Martin T
MT
Cabrera
Betty L
BL
Kolodner
Richard D
RD
Carethers
John M
JM
D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000494:pharmacology*; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics* / Q000494:pharmacology*; D020137:Base Pair Mismatch; D018736:DNA Adducts; D004260:DNA Repair; D045643:DNA Repair Enzymes; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000494:pharmacology*; D019008:Drug Resistance, Neoplasm; D005472:Fluorouracil / Q000494:pharmacology*; D006801:Humans; D018895:Microsatellite Repeats; D051719:MutS DNA Mismatch-Binding Protein; D009841:Oligonucleotides
D000964:Antimetabolites, Antineoplastic; D001426:Bacterial Proteins; D018736:DNA Adducts; D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D000251:Adenosine Triphosphatases; D051719:MutS DNA Mismatch-Binding Protein; D045643:DNA Repair Enzymes; D005472:Fluorouracil