The purification of a human mismatch-binding protein and identification of its associated ATPase and helicase activities.
A mismatch-binding protein has been purified an estimated 4500-fold from HeLa nuclear extracts using four different chromatographic steps. Two polypeptides of apparent molecular weight of 160,000 and 100,000 were present in the final affinity-purified fraction as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Partial proteolytic clipping of the protein-DNA complexes visualized after UV treatment indicated that the 100-kDa polypeptide is most likely a degradation product of the 160-kDa polypeptide. UV cross-linking experiments have shown that both these polypeptides bind specifically to oligonucleotide duplexes containing G/T mismatches. Direct DNA binding studies and band-shift competition assays showed that although the mismatch-binding protein binds with highest affinity to oligonucleotides containing G/T mismatches, it is also capable of binding to oligonucleotides containing other mispairs. The purified protein has an associated Mg(2+)-dependent ATPase activity, which is markedly enhanced in the presence of single-stranded DNA. A helicase capable of unwinding a 34-mer oligonucleotide, annealed to a complementary sequence in single-stranded M13, also copurified with the mismatch-binding protein. This reaction occurs in an ATP- and magnesium-dependent manner.
Hughes
M J
MJ
Istituto di Ricerche di Biologia Molecolare, Pomezia, Roma, Italy.
Jiricny
J
J
D000251:Adenosine Triphosphatases / Q000302:isolation & purification* / Q000378:metabolism*; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism*; D002846:Chromatography, Affinity; D004265:DNA Helicases / Q000302:isolation & purification* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D000251:Adenosine Triphosphatases; D004265:DNA Helicases