Mapping the minimal domain of hMSH-2 sufficient for binding mismatched oligonucleotides.
The human MSH-2 gene product is a member of a highly conserved family of proteins involved in post-replication mismatch repair. Germline mutations in this gene have been implicated in hereditary non-polyposis colorectal cancer (HNPCC). Alterations in the coding region of the hMSH-2 gene result in a mutator phenotype with marked instability of microsatellite sequences, indicative of a deficiency in DNA repair. We have previously shown that a region of high homology between MutS proteins of different species containing a nucleotide binding domain, is sufficient to bind DNA containing specific mismatched residues. In order to determine the minimal domain of hMSH-2 necessary for binding mismatch-containing oligonucleotides, deletion analysis of the C-terminal region was performed. We have constructed a 5' and 3' deletion series, expressed each deletion as a bacterial fusion protein and assessed it for ATPase activity and its ability to identify mismatch containing DNA. Here we demonstrate that a 585 bp fragment encoding 195 amino acids within the C-terminal domain of hMSH-2 is sufficient to bind to DNA containing mismatches.
Whitehouse
A
A
Molecular Medicine Unit, St. James's University Hospital, United Kingdom. msjaw@stjames.leeds.ac.uk
Deeble
J
J
Taylor
G R
GR
Guillou
P J
PJ
Phillips
S E
SE
Meredith
D M
DM
Markham
A F
AF
D000251:Adenosine Triphosphatases / Q000378:metabolism; D003001:Cloning, Molecular; D004260:DNA Repair; D004268:DNA-Binding Proteins; D006801:Humans; D051718:MutS Homolog 2 Protein; D016296:Mutagenesis; D009841:Oligonucleotides / Q000378:metabolism*; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D000251:Adenosine Triphosphatases; C497172:MSH2 protein, human; D051718:MutS Homolog 2 Protein