[Establishment of a hMSH2/hMSH6 protein interaction system and functional evaluation of hMSH2 gene missense mutations].
OBJECTIVE
To construct a hMSH2/hMSH6 protein interaction system, and to use it for evaluating missense mutations detected in hMSH2 gene.


METHODS
Recombinant 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.


RESULTS
Both 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.


CONCLUSIONS
A 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.
Jiangsu Key Laboratory of Molecular Medicine, Nanjing University School of Medicine, Nanjing, Jiangsu 210093, P. R. China. Email: wangyap@nju.edu.cn.
Ming
M
Zhu
Yi-mei
YM
Fan
Yan-bei
YB
Zhu
Ya-ping
YP
Wang
D020816:Amino Acid Motifs; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D051718:MutS Homolog 2 Protein / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020125:Mutation, Missense; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D004268:DNA-Binding Proteins; C094443:G-T mismatch-binding protein; C497172:MSH2 protein, human; D051718:MutS Homolog 2 Protein