A yeast two-hybrid assay provides a simple way to evaluate the vast majority of hMLH1 germ-line mutations.
Germ-line mutations in the hMLH1 gene are the most frequent cause of hereditary nonpolyposis colorectal cancer and are characterized by missense mutations at high frequency. We found a yeast two-hybrid assay to be an extremely useful and simple tool for evaluating the biological significance of such hMLH1 germ-line missense mutations; 78% (18 of 23) of the missense ones can be recognized as causative for nonpolyposis colorectal cancer. In addition, two of five variants not recognized as causative were thought to be rare polymorphisms. However, we could not detect any differences between wild-type hMLH1 and any of the nine already known polymorphisms causing amino acid alterations. Additional analysis demonstrated that the two-hybrid assay not only detected the dysfunctions at the COOH terminus of the hMLH1 protein necessary for the interaction with associated proteins but also detected a conformational change at the NH(2) terminus carrying ATPase activity. Thus, this method provides a simple and reliable system for accurate diagnosis of hMLH1 alterations.
Kondo
Emiko
E
Department of Molecular Pathology, Tohoku University School of Medicine, Miyagi 980-8575, Japan.
Suzuki
Hideyuki
H
Horii
Akira
A
Fukushige
Shinichi
S
D048868:Adaptor Proteins, Signal Transducing; D000230:Adenocarcinoma / Q000235:genetics*; D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D002352:Carrier Proteins; D018389:Codon, Nonsense / Q000235:genetics; D018388:Codon, Terminator / Q000235:genetics; D003123:Colorectal Neoplasms, Hereditary Nonpolyposis / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D024202:Electrophoretic Mobility Shift Assay; D016368:Frameshift Mutation; D015723:Gene Library; D018095:Germ-Line Mutation; D006801:Humans; D000070957:MutL Protein Homolog 1; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics*; D009687:Nuclear Proteins; D011110:Polymorphism, Genetic; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017384:Sequence Deletion; D020798:Two-Hybrid System Techniques
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018389:Codon, Nonsense; D018388:Codon, Terminator; C501486:MLH1 protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D000070957:MutL Protein Homolog 1