Figure 4

Nucleotide and DNA linking number dependence of compaction. (A) ATP and oligomeric state dependence on compaction. Summary of experiments testing the monomeric and multimeric forms of condensin in the presence and absence of ATP (1 mM). Each bar represents at least 4 independent trials (number of trials for each case are indicated by the number N on the bars). Significantly stronger compaction is obtained in the multimer + ATP case relative to the other cases (monomer + ATP, monomer-ATP, multimer-ATP), suggesting that only the condensin multimer is able to use ATP to drive enhanced DNA compaction. 1 mM magnesium chloride was present in each experiment. Data sets for each bar graph are given numerically and shown as scatter plots in Supplementary Worksheet 1. (B) Linking number and oligomeric state dependence on compaction. Summary of experiments testing the monomeric and multimeric forms of condensin in the presence of ATP plus magnesium (1 mM each) on DNA tethers of fixed values of linking number, as well for nicked DNA (zero DNA torsional stress). Each bar shows data from at least 4 independent trials. Essentially the same compaction is achieved for each monomer + ATP and multimer + ATP reactions for all cases of DNA torsional stress, indicating that torsional stress plays little role in the compaction reaction. Note that the Lk = 0 data are those shown in Fig. 4A. (C) Nucleotide effect on DNA compaction. Either monomeric or monomeric condensin was preincubated with 1 mM ATP, ATPγS, ADP or no nucleotide and used for a DNA compaction assay. each bar represents at least 4 independent trials. The enhanced compaction seen for the multimer + ATP case is absent for the cases of multimer combined with ATP or nonhydrolyzable ATPγS and for all monomer cases, indicating that the enhanced DNA compaction involves ATP hydrolysis. Note that the no-ATP and ATP data are those shown in Fig. 4A. (D) ATP incubation and DNA compaction. Multimeric condensin was used for a compaction assay where the condensin was either preincubated with 1 mM ATP, or where buffer plus ATP was flowed in exogenously (“flow-through”) after initial tether compaction with multimeric condensin in the absence of ATP. The significant enhancement of DNA compaction in the preincubated case is reduced in the flow-through case, indicating that presence of ATP at the beginning of the reaction is important to achieving strong DNA compaction. Note that the preincubated case data is the same as that for the multimer shown in Fig. 4A.