Abstract
Condensin, one of the most abundant components of mitotic chromosomes, is a conserved protein complex composed of two structural maintenance of chromosomes (SMC) subunits (SMC2- and SMC4-type) and three non-SMC subunits, and it plays an essential role in mitotic chromosome condensation. Purified condensin reconfigures DNA structure using energy provided by ATP hydrolysis. To know the regulation of condensin in somatic cells, the expression level, subcellular localization, and phosphorylation status of human condensin were examined during the cell cycle. The levels of condensin subunits were almost constant throughout the cell cycle, and the three non-SMC subunits were phosphorylated at specific sites in mitosis and dephosphorylated upon the completion of mitosis. Subcellular fractionation studies revealed that a proportion of condensin was tightly bound to mitotic chromosomes and that this form was phosphorylated at specific sites. Condensin purified from mitotic cells had much stronger supercoiling activity than that purified from interphase cells. These results suggest that condensin functions in somatic cells are regulated by phosphorylation in two ways during the cell cycle; the phosphorylation of specific sites correlates with the chromosomal targeting of condensin, and its biochemical activity is stimulated by phosphorylation.
Highlights
Chromatin undergoes extensive structural changes during the eukaryotic cell cycle
Protein Levels of Condensin Subunits Are Almost Constant through the Cell Cycle—Condensin is a five-subunit protein complex that plays an essential role in mitotic chromosome condensation
The five human condensin subunits were immunoprecipitated from whole cell extracts prepared under nondenaturing conditions by the anti-hCAP-E or anti-hCAP-G antibody, demonstrating that they associate with each other and form a complex in human cells similar to that observed in Xenopus egg extracts (Fig. 1B, lanes 1 and 3)
Summary
SDS buffer contains 50 mM Tris-HCl (pH 8.0), 0.6% SDS, and 1 mM dithiothreitol. Mild lysis buffer contained 0.1 M Na-Pi (pH 7.2), 20 mM EDTA, 0.5 M NaF, 1% Nonidet P-40, and 0.25 M NaCl. CSK buffer contained 10 mM PIPES (pH 6.8), 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol, 0.25 mM phenylmethylsulfonyl fluoride, 10 mM NaF, 0.1 mM ATP, and the Complete protease inhibitor mixture set (Roche Applied Science). XBE2-gly contains 10 mM HEPES (pH 7.7), 100 mM KCl, 5 mM EGTA, 2 mM MgCl2, 0.1 mM CaCl2, and 10% glycerol. Hypotonic buffer contains 20 mM HEPES (pH 7.0), 50 mM NaCl, 0.1 mM EDTA, 10% glycerol, 1 mM dithiothreitol, 0.25 mM phenylmethylsulfonyl fluoride, and the Complete protease inhibitor mixture set (Roche Applied Science)
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