oligomers. This result is consistent with previous observations of comparable NC binding to DNA and RNA molecules (49,50). Another very important result is that the binding constants of NC and Gag to these short oligonucleotides are very similar, see Table 3. This result is in contrast to 10-fold stronger binding of Gag to long dsDNA, observed in our stretching study (Table 1). One possible explanation for this discrepancy is that Gag binds the longer dsDNA by some additional region besides NC, most likely by the matrix domain of Gag (MA). The alternative explanation is that the longer nucleic acid molecule is capable of binding several Gag molecules simultaneously, while the short oligonucleotides used (10 nt) can likely accommodate only one Gag molecule. In this case, even if the individual Gag–DNA and NC–DNA binding constants are equally strong, Gag–Gag interactions can contribute to the stronger apparent Gag binding observed in DNA stretching experiments. In Discussion below we explore this hypothesis and estimate the DNA binding cooper- ativity parameter of Gag, and the corresponding free energy of Gag–Gag interaction. DISCUSSION In this work we used a single DNA molecule stretching tech- nique, as well as a tryptophan ﬂuorescence assay, in order to understand the speciﬁc features of the interaction with DNA of the HIV-1 NC protein while in the context of Gag as well as in its three differently processed forms. Our major goal is to see how the sequential proteolysis of HIV-1 NC affects its nucleic acid interaction properties, and whether or not this can be correlated with the change of NC’s function in various stages of the viral life cycle. While the NC protein within the context of Gag appears to have mostly a nucleic acid binding and packaging function, supporting Gag self-assembly into VLP, the processed forms of NC appear to act mostly as nucleic acid chaperones. The latter ﬁnding means that NC is capable of greatly facilitating nucleic acid restructuring processes that lead to the more stable annealed state of these molecules. While Gag facilitates tRNALys annealing to the 3 HIV-1 primer binding site (51–53), it has not been shown to facilitate more complex nucleic acid rearrangements, such as those involved in strand transfer. Until recently, it was not clear which properties of NC are responsible for its chaperone activity. However, recent studies of the kinetics of annealing (54), strand transfer (25,55–58) and recombina- tion (59–62) have shown that the chaperone activity of NC has two major components: helix destabilization and strand aggregation (63). HIV-1 NC is able to weakly destabilize all nucleic acid base pairs, allowing it to melt unstable nucleic acid inter- mediates, but preventing melting of the ﬁnal, stably annealed structures. Thus, melting by NC of the short helical regions bordered by mismatches or bulges provides the single- stranded nucleation sites for the new helices. In addition, NC-induced duplex destabilization accelerates various processes of strand exchange (63). On the molecular level, the capability of HIV-1 NC to destabilize nucleic acid duplexes relies on its preferential binding to single-stranded nucleic acids via the stacking of the aromatic residues of its zinc ﬁngers with unpaired bases (49,64–66). In our Nucleic Acids Research, 2006, Vol. 34, No. 2 601 force-induced melting studies, this duplex destabilizing activ- ity of NC is observed directly by measuring the changes in DNA base pair stability upon addition of NC. In the present work this duplex destabilizing activity of all three NC proteins studied appeared to be quite similar, with each base pair destabilized by processed NC proteins by 1 kcal/mol bp. Since the average stability of a base pair in the absence of NC was 1.4 kcal/mol in 50 mM NaCl, this is a signiﬁcant but incomplete destabilization induced by NC, which is con- sistent with the chaperone function of NC. Similar destabil- ization produced by all three proteins seems reasonable, taking into account the fact that they all share the same DNA binding domains. In contrast, according to our measurements, the DNA bind- ing constant of Gag is about 10-fold higher than that of the other three NC proteins. If the NC domain constitutes the main binding domain of Gag to DNA, the enhancement of the protein–DNA association constant comes most likely from an additional Gag–Gag interaction upon binding of the protein to DNA (i.e. cooperative binding) (30), although we cannot rule out additional interactions from other parts of Gag. In the limit of low protein binding, Q<<1, the fraction of protein bound Q, is proportional to the protein concentration c, as in Equation 2: Q ¼ Keff · c‚ Keff ¼ K · w and the effective binding constant includes the cooperativity parameter, w, deﬁned below: 5 6 7 w ¼ exp ðm=2Þ · dGGag Gag kBT Here dGGag Gag is the free energy of Gag–Gag interaction per protein molecule, m is the number of the nearest-neighbor Gag molecules when bound to DNA, the factor 1/2 takes into account that each interaction is shared by the two Gag molecules, and kBT is the thermal energy per statistical degree of freedom, which at room temperature is 0.6 kcal/mol. The fact that the effective binding constant of Gag is 10- fold higher than for either NC protein implies that w 10. This estimate, of course, assumes that the intrinsic binding constant of Gag to DNA, K, is the same as the binding constant of NC to DNA. By making this assumption and using Equation 7, we can estimate that m=2 · dGGag Gag ¼ kBT 1n w 2:3kBT ¼ 1:4 kcal=mol: 8 The pair-wise attraction free energy dGGag Gag can be estim- ated from the net free energy of cooperative interactions per Gag molecule given by Equation 8, for the particular number of nearest neighbors, m. The m range for Gag is likely from 3 to 4, with individual interacting subunits of capsid forming dimers of trivalent capsid molecules (67–69), each of which has 2–3 neighbors in addition to the dimer partner. In our experiments the number of interacting Gag neighbors bound to the single stretched DNA molecules could be as low as 2, given the fact that the DNA is stretched. We can therefore estimate the Gag–Gag interaction free energy to range between dGGag Gag ¼ 0.7 and 1.4 kcal/mol.