Smc4 ATPase mutations that compromise chromosome condensation exhibit reduced ATP hydrolysis in vitro.

<p>(A) ATPase activity of Smc4, Smc4-K191M, and the H-loop and C-helix mutants were compared using equal amounts of protein (1.5 μmol). Each condensin complex was incubated for 1 hour in ATPase buffer at 30 °C. ADP accumulation was measured with ADP-Glo luminescence kit (Promega). Error bars represent SEM (<i>N</i> ≥ 8 for all complexes). Star symbols signify the following <i>p</i> values: ** <i>p</i> < 0.01 and **** <i>p</i> < 0.0001. N.s. indicates no significant difference. (B) DNA binding to 60-nt ssDNA of condensin complexes used in (A) was analyzed using fluorescence anisotropy. Increasing amounts of each condensin complex were incubated with 10 nM 6-FAM ssDNA until the signal became saturated. The binding <i>K</i>_<i>d</i> was calculated using One site-Specific binding with Hill slope equation, and the 95% confidence interval for each complex is depicted in the figure (<i>N</i> = 3 for all complexes). See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003980#pbio.2003980.s007" target="_blank">S1 Data</a> for primary data. 6-FAM, 6-carboxyfluorescein; ssDNA, single-stranded DNA.