Mutational analysis of the nucleotide binding domain of the mismatch repair enzyme hMSH-2.
The genes involved in postreplicative DNA mismatch repair are a highly conserved family of proteins. In humans, germline mutations in these genes (hMSH-2, hMLH-1, hPMS-1, and hPMS-2) have been implicated in hereditary nonpolyposis colorectal cancer (HNPCC). We have previously shown that a region of high homology between the members of this class of proteins in different species contains a type A nucleotide binding site consensus sequence which has ATPase activity and is sufficient to bind DNA containing specific mismatched residues (1). To identify residues which are necessary for this activity, we have created a range of mutants containing amino acid substitutions within the nucleotide binding domain of hMSH-2. These mutants have been expressed and assessed for ATPase activity and their ability to identify mismatch-containing DNA. Here we demonstrate that a variant protein which has the conserved residue Lys 675 within the nucleotide binding consensus sequence altered to an alanine has severely impaired ATPase activity and is unable to bind DNA containing specific mismatched residues.
Whitehouse
A
A
Molecular Medicine Unit, St. James's University Hospital, Leeds, United Kingdom. whitehouse@stjames.leeds.ac.uk
Parmar
R
R
Deeble
J
J
Taylor
G R
GR
Phillips
S E
SE
Meredith
D M
DM
Markham
A F
AF
D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004260:DNA Repair / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D051718:MutS Homolog 2 Protein; D009711:Nucleotides / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D004268:DNA-Binding Proteins; D009711:Nucleotides; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D000251:Adenosine Triphosphatases; C497172:MSH2 protein, human; D051718:MutS Homolog 2 Protein