Dense packaging DNA in eukaryotic nuclei, sperm nuclei, viruses, and bacteria is necessary for proper cellular functioning. DNA assembly by multivalent ions is a critical testing ground for understanding not only in vivo compaction of DNA but also the physics of interactions between charged molecules. If a sufficient concentration of multivalent ions is present, DNA will spontaneously assemble into an ordered array. The helices do not collapse to touching but are rather separated by 0.5-1.5 nm of solvent depending on the nature of the condensing ion. Attractive and repulsive forces balance at the equilibrium spacing. By combining the osmotic stress, pushing experiments with single molecule, magnetic tweezers, pulling experiments, we were able to separate the attractive and repulsive free energies at the equilibrium spacing for the several commonly used condensing agents. The results confirmed our previous hypotheses for hydration forces. There is a 0.25 nm decay length exponential repulsive force that is the hydration equivalent of the image charge repulsive force in electrostatics. The hydration atmosphere extending from a solvated surface stabilizes water structuring at the surface. Disruption of the atmosphere by replacing water with another surface will lower hydration energies regardless of the water structuring on the other surface. Repulsion should depend predominately on the water structuring of groups on the DNA surface and perhaps on the mode of binding (phosphate backbone or grooves), but not on the correlations of these groups with apposing helices. The attraction is also characterized by an exponential force but with twice the decay length (0.5 nm) of the repulsive force. Attraction results from the direct interaction of surface hydration structures. Perturbations in water structure around one surface due to the close presence of another surface can either weaken or strengthen hydration energies depending on the mutual structuring water. We postulated that the attractive force had the same exponential 0.5 nm decay length as observed previously for repulsive hydration forces, but that the force was now attractive because of correlations in complementary water structuring on apposing helices. By further investigating biogenic alkylamines and arginine peptides of varying lengths, we determined that the magnitude of the attractive force increases with the number of charges consistent with a constant loss in entropy from correlating a single molecule regardless of charge, but a gain in interaction energy that increases with the number of charges. The repulsive force is only slightly dependent on the number of charges, as expected, but does depend on the chemical nature of the bound counterion. We are now investigating packaging forces of salmon protamine assembled DNA. Protamines are small, arginine-rich peptides used to package DNA in sperm heads. Protamine-DNA forces resemble polyarginine-DNA interactions. The equilibrium spacing between DNA helices with protamine, however, corresponds to tetra- or penta-arginine, not the 21 arginine charges actually present. We have separated protamine DNA forces into their attractive and repulsive components. The attractive force magnitude itself is very close to that expected for 21 arginine charges based on our previous measurements of arginine peptides. The repulsive force, however, is significantly larger than expected from the arginine peptide measurements, suggesting that the neutral amino acids present in protamine result in an increased residual repulsion. We have confirmed this hypothesis by measuring DNA force curves with synthetic hexa-arginines that incorporate increasing numbers of neutral amino acids We have also measured DNA assembly forces in salmon sperm nuclei. The in vivo sperm and in vitro, reconstituted DNA-salmon protamine forces are virtually identical. These force measurements thus have direct biological application. We can use our knowledge of DNA assembly forces to investigate the connection between DNA packaging in sperm heads, DNA damage, and male infertility. There are several reports in the literature correlating human male infertility with protamine abnormalities. Cumulative DNA damage due to reactive oxygen species is a major direct contributor to male infertility since DNA repair systems are absent in sperm. We hypothesize that incorrect assembly of DNA by insufficient or altered protamines will result in less tightly packaged DNA in sperm (a larger than normal spacing between helices) and, consequently, a greater potential exposure of DNA to oxidizing free radicals. We can test this hypothesis by direct measurements of the distance between helices in sperm of normal and infertile males and of the accumulated DNA damage.
Showing the most recent 10 out of 17 publications
