602 Nucleic Acids Research, 2006, Vol. 34, No. 2 These results suggest that the individual CA–CA interac- tions of HIV-1 Gag are much weaker than any typical func- tionally important protein–protein interaction. Unfortunately, other direct measurements of such weak interactions are not yet available for HIV-1 Gag. However, the cooperative Gag–DNA binding constant that we infer from our results, KGag ¼ 109 M 1 is in agreement with the typical values of the reciprocal critical concentration of capsid molecules required for the capsid self-assembly Kap ¼ 106–109 M 1 for several other viruses studied (70). Thus, cooperative bind- ing of Gag to DNA is consistent with its enhanced capability to bind to and package nucleic acids. However, multiple Gag– Gag interactions are expected to lead to a slowing of this protein’s motion on nucleic acid. This, in turn, should result in the interference of Gag with the fast nucleic acid duplex melting/annealing observed in the presence of NCp7, as reﬂec- ted in the signiﬁcant hysteresis observed in DNA stretching curves in the presence of Gag (Figure 5). Therefore, because of this slow unbinding (relative to NCp7) as well as the inability of Gag to destabilize the DNA duplex, the interactions of Gag with nucleic acids are not likely to be optimized for complex rearrangements of nucleic acid secondary structure such as those required for strand transfer. The second HIV-1 NC activity that contributes to its chaperone function is its ability to promote annealing of nucleic acid strands via their aggregation. This activity is especially important for facilitating the bimolecular nucleation step of strand annealing. The NC-induced rate enhancement of such reactions is especially signiﬁcant in solutions of low ionic strength, where it can reach up to 103–105-fold (54,71–73). This is because the NC protein facilitates the new duplex nucleation by screening the negative phosphate charges of the nucleic acid strands, just as is observed in high salt. However, the facilitating effect of HIV-1 NC on duplex nucleation goes far beyond this high salt effect (72). The additional rate enhancement comes from NC-induced nucleic acid–nucleic acid attraction (74,75). This electrostatic attraction is a phenomenon similar to the nucleic acid aggregation induced by multivalent cations (76–78). The attraction induces nonspeciﬁc aggregation of all nucleic acids, increasing the local concentration of strands, thus facilitating the diffusional search of the complementary regions for each other. The most important condition for this mechanism of nucleic acid annealing rate enhancement is that the nucleic acid molecules should remain highly mobile relative to each other within such an aggregate. These conditions seem to be fulﬁlled in the case of HIV-1 NCp7. Being a highly charged cationic molecule, NCp7 most likely produces the nucleic acid aggregation in a manner similar to that of other simple multivalent cations (76–82) via its electrostatic interaction with nucleic acids. This highly mobile and nonspeciﬁc nucleic acid aggregation induced by NCp7 can be contrasted with the much stronger and much less mobile ‘sticking’ of nucleic acid molecules produced by the cooperative association of the hydrophobic parts of the nucleic acid binding proteins, such as Gag. This concept is reﬂected in the stretching and relaxation curves shown in Figure 5, which in the cases of NCp7, NCp15, NCp9 and Gag proteins show progressively larger hysteresis. There are several factors that may cause the hysteresis to increase in the presence of typical DNA binding proteins. First of all, a protein that binds preferentially to ssDNA, as NC does, may be slow to unbind, and therefore might interfere with strand annealing (40,83). Moreover, in order for the relaxation curve to coincide with the stretching curve, the fraction of the base pairs annealed should be in true equilibrium. This means that on the time scale of a single step, i.e. 1 s, in which the force is being averaged in our experiment, multiple events of melting and reannealing of the base pairs should occur, such that the fraction of the annealing and melting base pairs aver- ages to the thermodynamic equilibrium quantity. This, in turn, implies that many protein molecules bound to the 1000 bp long piece of DNA that is melted in each step should be able to unbind ssDNA and rebind dsDNA many times on the time scale of 1 s. This is a rather stringent requirement on the ability of the protein to adjust to the state of DNA. Based on the experimental data showing very small hysteresis in the pres- ence of NCp7, we argue here that NCp7 is indeed such a very ‘fast’ binding and unbinding protein. This hypothesis is in good agreement with the very high mobility of NCp7 when bound to various nucleic acid molecules, as observed in NMR experiments. Indeed, NMR studies have shown that the majority of NCp7 molecules, when bound to nucleic acids, are able to rapidly switch between numerous possible conformations, which are aver- aged out on the millisecond time scale of an NMR experiment (84–86). Also, recent ﬂuorescent energy transfer studies of the TAR DNA molecule have shown that the NCp7 (12–55) fragment is able to facilitate the rate of the terminal DNA duplex opening and closing on the 10 4–10 3 s time scale by several-fold (87–89). This implies that NCp7, lacking the basic N-terminal tail, is capable of changing its binding mode between the ssDNA- and dsDNA-bound modes even faster than that. This is not impossible, since in order to adjust to a new state of DNA, the protein does not have to completely unbind and rebind the nucleic acid. Instead, it may simply change the conformation of its aromatic residues between DNA bases, i.e. stacked and unstacked, in addition to undergoing other small conformational changes. Such move- ments can easily occur on the microsecond time scale. It is likely that protein–protein interactions resulting in cooperative protein binding to DNA are responsible for the observed hysteresis in the presence of Gag. The Gag–Gag interactions estimated from the high value of the Gag–DNA binding constant above are consistent with the large hysteresis in the presence of high concentrations of Gag (1–10 nM). Similar features are present for NCp9 and NCp15 proteins, which induce hysteresis that is much stronger than in the case of NCp7. This ﬁnding correlates with the results of Khan and Giedroc (72), who reported that, in contrast to the com- pletely processed NCp7, NCp9 shows moderate DNA binding cooperativity. In addition, these authors found that while NCp7 binds and unbinds DNA very fast on the time scale of a Trp ﬂuorescence quenching measurement, the NCp9 protein was shown to undergo some slow changes in its bind- ing to DNA (72). Taken together, these ﬁndings suggest that the distal domains in the partially processed NC protein and, particularly, the unstructured, ﬂexible and highly hydrophobic p1 sequence present in NCp9, may participate in protein–protein contacts. Thus, the hysteresis and possible DNA–protein binding cooperativity that are observed most strongly for Gag and