Characterization of mutant MUTYH proteins associated with familial colorectal cancer.
OBJECTIVE
The human mutyh gene encodes a base excision repair protein that prevents G:C to T:A transversions in DNA. Biallelic mutations in this gene are associated with recessively inherited familial colorectal cancer. The aim of this study was to characterize the functional activity of mutant-MUTYH and single-nucleotide polymorphism (SNP)-MUTYH proteins involving familial colorectal cancer.


METHODS
MUTYH variants were cloned and assayed for their glycosylase and DNA binding activities using synthetic double-stranded oligonucleotide substrates by analyzing cleavage products by polyacrylamide gel electrophoresis.


RESULTS
In this study, we have characterized 9 missense/frameshift mutants and 2 SNPs for their DNA binding and repair activity in vitro. Two missense mutants (R260Q and G382D) were found to be partially active in both glycosylase and DNA binding, whereas 3 other missense mutants (Y165C, R231H, and P281L) were severely defective in both activities. All of the frameshift mutants (Y90X, Q377X, E466X, and 1103delC) were completely devoid of both glycosylase and DNA binding activities. One SNP (V22M) showed the same activity as wild-type MUTYH protein, but the other SNP (Q324H) was partially impaired in adenine removal.


CONCLUSIONS
This study of MUTYH mutants suggests that certain SNPs may be as partially dysfunctional in base excision repair as missense-MUTYH mutants and lead to colorectal carcinogenesis.
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
Mohsin
M
Ali
Hyeja
H
Kim
Sean
S
Cleary
Claire
C
Cupples
Steven
S
Gallinger
Robert
R
Bristow
D000225:Adenine / Q000378:metabolism; D011125:Adenomatous Polyposis Coli / Q000201:enzymology / Q000235:genetics*; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D015179:Colorectal Neoplasms / Q000201:enzymology / Q000235:genetics*; D004247:DNA / Q000378:metabolism; D045647:DNA Glycosylases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004260:DNA Repair; D004591:Electrophoresis, Polyacrylamide Gel; D016368:Frameshift Mutation; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D020125:Mutation, Missense; D020641:Polymorphism, Single Nucleotide
D004247:DNA; D045647:DNA Glycosylases; C061738:mutY adenine glycosylase; D000225:Adenine