Protamines are small, arginine-rich, nuclear proteins that condense the spermatid genome into a genetically inactive state. Unlike histone compacted DNA, the physical properties of reconstituted of protamine - DNA assemblies closely resemble those of DNA condensed by multivalent cations that are much smaller, have much less charge, and have been better characterized such as divalent manganese, cobalt hexammine, spermidine, spermine, and several arginine oligopeptides. DNA is packaged by protamine to densities within the range seen for DNA condensed with the smaller multivalent ions. More importantly, the forces measured for reconstituted salmon protamine DNA arrays by the osmotic stress technique coupled with x-ray diffraction show very similar characteristics to DNA precipitated by multivalent ions. Additionally, we have also shown that reconstituted salmon protamine DNA arrays and native salmon nuclei give the same osmotic stress force curves. We hypothesize that male infertility from protamine deficiency and errors in modification is due to looser packing of DNA in sperm nuclei than is optimal and hence greater accessibility to mutagens and oxidizing species. Upon condensation with the smaller multivalent ions or protamine, the resulting compacted structures have well-defined equilibrium separations of the DNA double helices of 0.7 1.5 nm, depending on the identity of the condensing ion. The finite separation of helices indicates a delicate balancing of a short ranged repulsive force with a longer ranged attraction. The physical origins of the forces acting to compact multivalent ion - DNA assemblies are still debated. We have argued for water-structuring or hydration forces on the basis of our previous extensive measurements of forces between both charged and uncharged molecules. Most theories of DNA assembly include some form of correlation of positive charges on one helix with negative phosphate charges on another in order to account for attraction. Using combined osmotic stress and single molecule tweezing experiments, we have previously characterized the distance dependence of the two component forces. The attractive force varies with DNA-DNA spacing as a 0.4 0.5 nm decay length exponential;whereas the repulsive force is a 0.2 0.25 nm decay length exponential. The factor of two difference in decay lengths suggests that the attractive force results from a direct interaction of charged groups on apposing helices;whereas the repulsive force is an image-charge or its hydration equivalent force. Using this constraint, we can effectively separate the attractive and repulsive contributions to the force - distance curves. Using synthetic peptides we were able to elucidate several features of protamine compaction of DNA. All vertebrate protamines favor arginines almost exclusively over lysines. We found that arginine peptides can assemble DNA to much tighter spacings than lysine peptides of the same length. Packaging helices with protamines based on lysines in sperm nuclei would leave the DNA more vulnerable to damage. We hypothesize that differences in binding geometry between the two amino acids affect the correlation of charges on apposing helices. The incorporation of neutral amino acids into arginine peptides increases the amplitude of the short-ranged repulsive force while affecting attraction only slightly. We hypothesize that these neutral amino acids increase the image charge like repulsive force by displacing additional water from DNA grooves. The amplitudes of the repulsive and attractive forces for salmon protamine-DNA complexes can be quantitatively predicted from the number of arginines and the fraction of neutral amino acids. The incorporation of one negatively charged aspartate or phosphorylated serine into a hexa-arginine peptide increases the amplitude of the short-ranged repulsion dramatically, the equivalent of incorporating 8 neutral amino acids. The longer ranged attraction is moderately decreased, but is consistent with a net +5 charge. This likely has biological implications for spermatogenesis. The replacement of histones by protamines occurs in several steps. First acetylated histones are replaced by transition proteins. Serine phosphorylated protamines than replace the transition proteins. It is only after removing the phosphate groups that DNA is tightly packed. Incomplete dephosphorylation has been suggested a cause of increased DNA damage and male infertility. The results here show that phosphorylation has a much larger effect on weakening the net attraction between helices than simply reducing the protamine charge would suggest. We have fractionated salmon sperm nuclei on a sucrose gradient and measured x-ray spacings between DNA helices. More slowly sedimenting nuclei are characterized by a larger average spacing between helices than for denser fractions. Careful analysis of the scattering peaks indicates that the increased average spacing is the result of an increased spread of interhelical distances from normal to larger distances rather than a simple shift of a scattering peak with constant width to larger spacings. There are several differences between mammalian and piscine protamines. Many mammals have two protamines, P1 and P2, both of which are longer than piscine protamines. The average fraction arginine of mammalian protamines, 50-60%, is significantly smaller than for fish, 65-70%. On the basis of our results, the increase in neutral amino acid content would be expected to decrease the net attraction, significantly increasing the equilibrium spacing between helices in mammalian sperm. A looser packaging of DNA due to an increased fraction of neutral amino acids would increase accessibility of mutagens and reactive oxidizing species to the DNA causing increased damage. This might explain another difference between mammalian and piscine protamine-DNA packaging. Extensive disulfide bridges between protamines are present in mammalian sperm. Most piscine protamines, on the other hand, do not have cysteines. The disulfide bridges in mammals may be required for tight DNA packaging, overcoming the increased fraction of neutral amino acids. We are now investigating DNA packaging in bull sperm. Bulls only have a P1 protamine and represent an intermediate in complexity between fish and humans sperm. The osmotic stress force curve for bull sperm nuclei with intact disulfide bonds is remarkably similar to that for salmon sperm nuclei. Reduction of the disulfide bonds with DTT leads to dramatic swelling of the nuclei (at least 10-fold in volume) and complete loss of x-ray order. The disulfide bonds are absolutely essential for dense packing of DNA. The osmotic stress force curve for reduced bull nuclei gradually merges at higher pressures with the unmodified bull sperm nuclei curve. Disulfide bonds do not reform if the DTT is removed from greatly swollen nuclei, but will reform if the DTT is removed and nuclei are compacted with osmotic stress. Future work includes the isolation of bull sperm protamine for reconstitution studies and density fractionation and x-ray analysis of bull sperm nuclei coupled with determination of 8-oxoguanine by mass spectrometry as an indicator of oxidative DNA damage.
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