The biochemical characterization of the DNA binding activity of pKi67.
Ki67 is only expressed in the nucleus of cycling cells. While it is employed as an operational marker of proliferation, little is known of the biochemical properties of this large protein. Using an immunoaffinity strategy for purification of pKi67, this study has shown that it can form higher-order complexes and can bind to DNA cellulose in vitro. No other co-purifying proteins could be identified, strongly suggesting that the DNA binding activity is an inherent property of pKi67. Using an electromobility shift assay, the affinity of pKi67 was shown using a range of different forms of DNA as competitors. Single-stranded DNA was the poorest competitor, followed by double-stranded DNA, with supercoiled DNA being the best competitor. In addition, it was found that purified pKi67 has a preference for AT-rich DNA. The DNA binding domain is mapped to the C-terminal domain of pKi67, and recombinant protein from the terminal 321 residues of pKi67 can bind DNA in vitro. GFP constructs from this domain were used to map regions that could target nucleolar localization and allow DNA binding. Finally, it was found that over-expression of the C-terminal 321 residues in cells induced chromatin disruption and apoptosis. These data provide strong evidence that pKi67 has a novel DNA binding activity within the C-terminal domain and that this protein can influence chromatin structure.
MacCallum
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DE
Department of Molecular and Cellular Pathology, University of Dundee, Ninewells Hospital and Medical School, UK.
Hall
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PA
D017209:Apoptosis / Q000235:genetics; D001667:Binding, Competitive; D002843:Chromatin / Q000235:genetics; D004278:DNA, Superhelical / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D019394:Ki-67 Antigen / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics; D013329:Structure-Activity Relationship
D002843:Chromatin; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D019394:Ki-67 Antigen; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins