Isolation of MutSbeta from human cells and comparison of the mismatch repair specificities of MutSbeta and MutSalpha.
A human MSH2-human MSH3 (hMSH2.hMSH3) complex of approximately 1:1 stoichiometry (human MutSbeta (hMutSbeta)) has been demonstrated in several human tumor cell lines and purified to near homogeneity. In vitro, hMutSbeta supports the efficient repair of insertion/deletion (I/D) heterologies of 2-8 nucleotides, is weakly active on a single-nucleotide I/D mispair, and is not detectably active on the eight base-base mismatches. Human MutSalpha (hMutSalpha), a heterodimer of hMSH2 and hMSH6, efficiently supports the repair of single-nucleotide I/D mismatches, base-base mispairs, and all substrates tested that were repaired by hMutSbeta. Thus, the repair specificities of hMutSalpha and hMutSbeta are redundant with respect to the repair of I/D heterologies of 2-8 nucleotides. The hMutSalpha level in repair-proficient HeLa cells (1.5 microg/mg nuclear extract) is approximately 10 times that of hMutSbeta. In HCT-15 colorectal tumor cells, which do not contain hMSH6 and consequently lack hMutSalpha, the hMutSbeta level is elevated severalfold relative to that in HeLa cells and is responsible for the repair of I/D mismatches that has been observed in this cell line. LoVo tumor cells, which are genetically deficient in hMSH2, lack both hMutSalpha and hMutSbeta, and hMSH3 and hMSH6 levels are less than 4% of those found in repair-proficient cells. Coupled with previous findings (J. T. Drummond, J. Genschel, E. Wolf, and P. Modrich (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10144-10149), these results suggest that hMSH2 partitions between available pools of hMSH3 and hMSH6 and indicate that hMSH2 positively modulates hMSH6 and hMSH3 levels, perhaps by stabilization of the polypeptides upon heterodimer formation.
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
J
J
Genschel
S J
SJ
Littman
J T
JT
Drummond
P
P
Modrich
D000251:Adenosine Triphosphatases; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D001482:Base Composition / Q000235:genetics; D004260:DNA Repair / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism; D029968:Escherichia coli Proteins; D005656:Fungal Proteins / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D051719:MutS DNA Mismatch-Binding Protein; D051718:MutS Homolog 2 Protein; D009687:Nuclear Proteins / Q000032:analysis; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D005656:Fungal Proteins; C094443:G-T mismatch-binding protein; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D000251:Adenosine Triphosphatases; C497172:MSH2 protein, human; D051719:MutS DNA Mismatch-Binding Protein; D051718:MutS Homolog 2 Protein; C497175:MutS protein, E coli