Experiments were conducted on positively supercoiled DNA bearing a CTP-less cassette.
a, UvrA alone compacts undamaged, supercoiled DNA in a non-specific manner even at concentrations as low as 10 pM. Trace shown obtained with 1 mM ATP; the same phenomenon is observed in the absence of ATP (data not shown). b, UvrB prevents non-specific interaction of UvrA with DNA; ( t = 0 s) 250 nM UvrB alone does not compact DNA, although it transiently interacts non-specifically and briefly with DNA in the presence of 1 mM ATP (see c– f). The same phenomenon is observed in the absence of ATP (data not shown); ( t = 2,000 s) addition of UvrB also prevents UvrA from compacting DNA non-specifically. On the basis of these data we set the working UvrB concentration to 250 nM: our measurements with UvrA can thus go up to 100 pM, which remains more than 90% saturated by this concentration of UvrB as shown by the fact that we can perform measurements without DNA compaction. c, d, Time-traces obtained on positively supercoiled DNA in the presence of 250 nM UvrB and 1 mM ATP show supercoiling-dependence of the dwell time ( t dwell) of UvrB-DNA ‘wrapping’ events. Indeed the amplitude of these events (~50–100 nm) is consistent with titration of a large positive supercoil by formation of a tight/compact, positive wrap of DNA around UvrB as observed in AFM imaging . 27 e, f, Histograms of the dwell time of the wrap state obtained above are fitted to single-exponential distributions, with a mean dwell time of ( e) 28 ± 2 s (s.e.m., n = 175, +5 turns), and ( f) 66 ± 5 s (s.e.m., n = 117, +6 turns). By performing experiments with no more than 250 nM UvrB and with only +4 turns of positive supercoiling, this wrap state is of order 10 s and does not significantly interfere with detection of Mfd–RNAP intermediates or their resolution, and UvrB safely inhibits DNA compaction activity by UvrA.