Establishment of a hMSH2/hMSH6 protein interaction system and functional evaluation of hMSH2 gene missense mutations.
- Author:
Ming ZHU
1
;
Yi-mei FAN
;
Yan-bei ZHU
;
Ya-ping WANG
Author Information
- Publication Type:Journal Article
- MeSH: Amino Acid Motifs; Base Sequence; DNA-Binding Proteins; chemistry; genetics; metabolism; Humans; Molecular Sequence Data; MutS Homolog 2 Protein; chemistry; genetics; metabolism; Mutation, Missense; Protein Binding; Saccharomyces cerevisiae; genetics; metabolism; Two-Hybrid System Techniques
- From: Chinese Journal of Medical Genetics 2013;30(5):559-564
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo construct a hMSH2/hMSH6 protein interaction system, and to use it for evaluating missense mutations detected in hMSH2 gene.
METHODSRecombinant plasmids pGADT7-hMSH2, pGBKT7-hMSH6 and 7 recombinant pGBKT7 plasmids with different hMSH6 domains were constructed through genetic engineering. Subsequently, site-directed mutagenesis was used to construct 10 mutant pGADT7-hMSH2 plasmids, which were transformed into yeast AH109. The growth of transformants was observed on a histidine-deficient culture.
RESULTSBoth hMSH6 MutSII-V and MutSIII-V could interact with hMSH2 in yeast AH109. Yeast two-hybrid transformants pGADT7-hMSH2/pGBKT7-hMSH6 MutSII-V were used to construct a hMSH2/hMSH6 protein interaction system. Compared with wild-type hMSH2, yeast two-hybrid transformants c.505A>G, c.1168C>T, c.1255C>A, c.1261C>A could grow normally, c.1223A>G, c.1886A>G, c.2108C>A and c.2516A>G grew slowly, c.518T>G and c.1664 delA could not grow in a histidine-deficient medium in yeast AH109.
CONCLUSIONA hMSH2/hMSH6 protein interaction system has been constructed with yeast two-hybrid system, which has been used for functional evaluation of hMSH2 gene missense mutations. c.518T>G is a pathological mutation. c.1223A>G, c.1886A>G, c.2108C>A, c.2516A>G may in part affect the hMSH2 function. And c.505A>G, c.1168C>T, c.1255C>A, c.1261C>A were innocuous variants.
