Characterization of the mismatch repair defect in the human lymphoblastoid MT1 cells.
Mutations in mismatch repair (MMR) genes predispose to hereditary nonpolyposis colon cancer. Those leading to truncated proteins bring about a MMR defect, but phenotypes of missense mutations are harder to predict especially if they do not affect conserved residues. Several systems capable of predicting the phenotypes of MMR missense mutations were described. We deployed one of these to study the MMR defect in MT1 cells, which carry mutations in both alleles of the hMSH6 gene. In one, an A-->T transversion brings about an Asp(1213)Val amino acid change in the highly conserved ATP binding site, whereas the other carries a G-->A transition, which brings about a Val(1260)Ile change at a nonconserved site. The hMSH2/hMSH6 (hMutS alpha) heterodimers carrying these mutations were expressed in the baculovirus system and tested in in vitro MMR assays. As anticipated, the Asp(1213)Val mutation inactivated MMR by disabling the variant hMutS alpha from translocating along the DNA. In contrast, the recombinant Val(1260)Ile variant displayed wild-type activity. Interestingly, partial proteolytic analysis showed that this heterodimer was absent from MT1 extracts, although both hMSH6 alleles in MT1 cells could be shown to be transcribed with an efficiency similar to each other and to that seen in control cells. The MMR defect in MT1 cells is thus the compound result of one mutation that inactivates the ATPase function of hMutS alpha and a second mutation that apparently destabilizes the Val(1260)Ile hMSH6 protein in human cells in vivo.
Szadkowski
Marta
M
Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland.
Iaccarino
Ingram
I
Heinimann
Karl
K
Marra
Giancarlo
G
Jiricny
Josef
J
D000483:Alleles; D000595:Amino Acid Sequence; D016367:Baculoviridae / Q000235:genetics; D020137:Base Pair Mismatch / Q000235:genetics*; D002460:Cell Line; D004260:DNA Repair / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008214:Lymphocytes / Q000166:cytology / Q000502:physiology; D008969:Molecular Sequence Data; D051718:MutS Homolog 2 Protein; D016297:Mutagenesis, Site-Directed; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D004268:DNA-Binding Proteins; C094443:G-T mismatch-binding protein; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; C497172:MSH2 protein, human; D051718:MutS Homolog 2 Protein