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 well characterized by us such as divalent manganese, cobalt hexammine, spermidine, spermine, and several arginine and lysine peptides. The forces measured for compacting salmon sperm nuclei, reconstituted salmon protamine DNA arrays, and bull sperm nuclei using the osmotic stress technique coupled with x-ray diffraction show very similar characteristics to DNA precipitated by multivalent ions. The equilibrium surface separations of the DNA double helices compacted with the smaller multivalent ions or protamines are well-defined and vary between 0.7 and 1.8 nm, depending on the condensing ion. The finite separation of helices indicates a balancing of short ranged repulsive forces with longer ranged attractions. We have previously characterized the distance dependence of the two component forces. The attractive force varies with DNA-DNA spacing as a 0.5 nm decay length exponential;whereas the repulsive force is a 0.25 nm decay length exponential. The factor of two difference in decay lengths indicates 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. Although the physical origins of the forces 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. Since DNA damage in sperm by oxidation and resultant fertility problems due to protamine deficiency and errors in modification are likely sensitive to DNA packing densities, we have systematically investigated the contributions of protamine amino acid composition to the component attractive and repulsive forces. 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 DNA with protamines based on lysines in sperm nuclei would leave the DNA more vulnerable to damage. Using synthetic arginine homopeptides of different lengths, we found that the attractive force amplitude increases with the inverse of the number of arginines, but that the shorter ranged repulsive force is insensitive to peptide length. Also using synthetic peptides we have quantitated the effect of incorporating neutral amino acids into hexa-arginine peptides. The fraction of arginines in native protamines varies from 50-55% for mammals to 65-70% for fish. As the fraction of arginine is decreased in synthetic peptides, the amplitude of the short-ranged repulsive force increases while the attraction force is affected only slightly. We hypothesize that these neutral amino acids increase the image charge like repulsive force by displacing water from DNA grooves. With the synthetic peptide data, the packing density of DNA in salmon sperm nuclei can be quantitatively predicted from the number of arginines and the fraction of neutral amino acids. On the basis of our results, the difference in the fraction arginine between mammalian and piscine protamines should result in a significantly larger spacing between helices in mammalian sperm compared to fish. A looser packaging of DNA due to a decreased arginine fraction would increase accessibility of ROS to the DNA. In spite of the difference in arginine content, however, the equilibrium spacing between DNA helices in bull sperm nuclei is practically the same as in salmon sperm. This is explained by the another major difference between mammalian and piscine protamines. Extensive intra- and inter-protamine disulfide bridges are present in mammalian sperm, but absent with piscine sperm nuclei. Reduction of bull sperm inter-protamine disulfide bridges with 1 mM DTT leads to a 30% increase in the surface-to-surface separation of DNA helices or a 70% increase in the volume accessible to ROS. The increased spacing is consistent with the expectation from the decreased arginine fraction of bull protamine without inter-protamine disulfide bonds. Disulfide bridges in mammals are required for tight DNA packaging, overcoming the decreased fraction arginine. Significantly, disulfide bonds are not reformed if reducing DTT is removed, but will reform if nuclei are compacted with osmotic stress. This then begs the question of how disulfide bonds are formed in sperm. The spacing between helices is still very tight. Reduction of both inter- and intra-protamine disulfide bonds with 10 mM DTT leads to dramatic swelling of the nuclei (at least 10-fold in volume) and complete loss of x-ray order. It has been found by others that decondensation of sperm nuclei in fertilized eggs requires glutathione reduction. It has found by others that the lighter fractions of density fractionated sperm have more DNA damage than the heavier fractions. This is the basis for using density fractionation of sperm as a standard practice in artificial reproduction technologies. We have fractionated salmon sperm nuclei on sucrose gradients and measured x-ray spacings between DNA helices. More slowly sedimenting nuclei do have a larger average spacing between helices than the 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 the scattering peak with constant width to larger spacings. This is the first time that it has been shown that there is a distribution of DNA packing densities in sperm nuclei. The addition of excess salmon protamine to the nuclei results in compaction. At least part of the poor packaging of the lighter density fractions is due to insufficient protamine. We have investigated the dependence of interhelical spacing on protamine-to-DNA ratio in reconstituted assemblies. Our results indicate that the lighter density sperm nuclei fractions have only 90% of the protamine necessary for normal packing densities. We are still optimizing an assay for direct measurement of protamine/DNA ratios in sperm nuclei. We have also found that salmon sperm nuclei also contain a small (1-2%) fraction of residual phosphorylated protamine. The replacement of histones by protamines occurs in several steps. Protamines are initially serine phosphorylated as they bind DNA. It is only after removing the phosphate groups that DNA becomes tightly packed. We have evidence that serine phosphorylation of protamines has a profound effect on DNA packing. The incorporation of one phosphorylated serine into a hexa-arginine peptide increases the amplitude of the short-ranged repulsion dramatically, the equivalent of decreasing the apparent arginine fraction to 40-45%. We are now investigating if the less dense salmon sperm nuclei fractions have more residual protamine phosphorylation than average. We are also in the process of isolating enough phosphorylated protamine to reconstitute DNA assemblies and measure DNA spacings by x-ray. Future work includes: (1) coupling the structural measurements with measurements of oxidative DNA damage;(2) density fractionation and x-ray analysis of bull sperm nuclei;and (3) further characterization of disulfide bonds in bull sperm nuclei in particular their reformation after DTT reduction.
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