Figure 5. Stretching (solid lines) and relaxing (dashed lines) curves for l-DNA in the presence of the NC proteins or Gag: (A) NCp7; (B) NCp9; (C) NCp15; (D) Gag. Protein concentrations are indicated in the figure. Data were collected in FIM buffer (see Materials and Methods) at room temperature. The stretching and relaxing curves in the absence of protein are also shown (black solid line and filled diamonds, respectively). the low pulling rates used here) of the melting forces in these experiments. Representative DNA stretching and relaxation curves for all four proteins are shown in Figure 5. The stretching portion Nucleic Acids Research, 2006, Vol. 34, No. 2 599 of the curve is used to obtain the DNA melting free energy per base pair in the presence of each protein, calculated at saturated protein binding. The results of this calculation are presented in Table 1. The addition of saturating amounts of any of the three NC proteins (20 nM) results in a low DNA helix stability of 0.35 ± 0.03 kcal/mol bp. This value is about 1 kcal/mol bp smaller than in the absence of protein at the 50 mM ionic strength used. The signiﬁcant duplex des- tabilization induced by NC is the result of the preferential interaction between its zinc ﬁngers and the single-stranded nucleic acids. In addition, NC produces some duplex stabil- ization due to increased electrostatic screening of DNA phos- phate charges. The latter effect depends on the ionic strength of the buffer used and results in a salt dependence of the total NC-induced duplex destabilization. Thus, at higher salt, the net NC-induced DNA destabilization can become slightly larger than 1 kcal/mol bp. The effect of Gag on duplex stability is less signiﬁcant, i.e. the free energy of base pair melting is only 0.3 kcal/mol bp lower than in the absence of NC, due to the overall increase in the melting force and small change in the transition width (Figure 5). One of the possible reasons why Gag does not signiﬁcantly destabilize the DNA duplex could be that its NC domain is not capable of interacting with the ssDNA bases via its zinc ﬁngers as efﬁciently as in the processed NC. This possibility, however, is contradicted by the results of our direct measurement of NC and Gag binding to short ss oligonucleotides, discussed below, showing ident- ical NC and Gag binding. The alternative explanation is that the binding of Gag molecules to dsDNA is always accom- panied by strong interaction between the neighboring Gag molecules. The latter may produce a torsional constraint on the local unwinding of dsDNA, thus stabilizing it relative to the unwound ssDNA. This hypothesis is supported by our observation of the increasing force at the midpoint of the DNA force-induced melting transition upon Gag binding. Such a strong increase in the DNA melting force was previ- ously observed when the stretched dsDNA was torsionally constrained (44). Thus, our results argue that the Gag poly- protein is inefﬁcient as a duplex destabilizer, in contrast to all processed forms of NC. This ﬁnding suggests that Gag should be much less efﬁcient at complex nucleic acid secondary structure rearrangements than the processed NC proteins. Figure 5 also shows DNA relaxation curves for all four proteins, all of which show different amounts of hysteresis (i.e. disparity between stretch and relax curves). We have also studied the complete stretch–relax cycles of l-DNA in the presence of many other retroviral NC proteins (M. Cruceanu, K. Stewart, R.J. Gorelick, K. Musier-Forsyth, I. Rouzina, M.C. Williams, manuscript in preparation), as well as of sev- eral mutants of HIV-1 NCp7 (M. Cruceanu, M.-N. Vo, R.J. Gorelick, K. Musier-Forsyth, I. Rouzina, M.C. Williams, manuscript in preparation), and the ORF1 protein from mouse LINE-1 retrotransposon (38). Among all DNA binding pro- teins studied using force-induced melting of DNA, including the proteins presented herein, HIV-1 NCp7 protein produces the smallest hysteresis of the DNA stretch–relax cycle. As shown in Figure 5, the hysteresis induced by the proteins studied here, at saturation, increased in the following order: HIV-1 NCp7, NCp15, NCp9 and Gag. However, while the hysteresis contains rich information on protein–DNA interactions, its interpretation is complex, and has never