Assembly Forces And Recognition Reactions

装配力和识别反应

• 批准号: 8553904
• 负责人: Donald Rau
• 金额: $ 32.37万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8553904
• 关键词: Accounting; Affect; Amino Acids; Arginine; Aspartate; Bacteriophages; Binding; Biological; Cations; Cell Nucleus; Cell physiology; Cellular biology; Characteristics; Charge; Chromatin; Complex; Congenital Abnormality; Coupled; Coupling; Cysteine; DNA; DNA Damage; DNA Packaging; DNA Repair; DNA delivery; Dependence; Disease; Disulfides; Equilibrium; Fishes; Fractionation; Future; Genetic; Genome; Goals; Histones; Human; Hydration status; Image; Infertility; Ions; Knowledge; Left; Length; Link; Lysine; Male Infertility; Mammals; Manganese; Mass Spectrum Analysis; Measurement; Measures; Modification; Mutagens; Neutral Amino Acids; Nuclear Proteins; Oligopeptides; Peptides; Phosphorylation; Physical condensation; Physics; Protamines; Protein Dephosphorylation; Proteins; Reaction; Salmon; Serine; Solutions; Specificity; Spermatids; Spermatogenesis; Spermidine; Spermine; Spontaneous abortion; Stress; Structure; Sucrose; Swelling; Techniques; Water; Width; Work; aqueous; base; density; design; disulfide bond; disulfide bond reduction; hexaamminecobalt(II); inorganic phosphate; macromolecule; nucleic acid structure; oxidative DNA damage; physical property; pressure; reconstitution; research study; single molecule; sperm cell; structural biology; synthetic peptide; theories

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.

精蛋白是小精氨酸，富含精氨酸的核蛋白，可将精子基因组浓缩到遗传活性状态。与组蛋白压实的DNA不同，精蛋白重新组成的物理特性 - DNA组件非常类似于由多价阳离子浓缩的DNA的物理特性，这些阳离子的浓缩阳离子较小，电荷要少得多，并且已经更好地表征了，例如甲状腺素，钴胺，己糖，精子，精子，精子，精子，精子，精子，精子和几种精氨酸寡肽。 DNA被精蛋白包装到与较小的多价离子凝结的DNA范围内的密度。更重要的是，通过渗透应力技术与X射线衍射相结合的重构鲑鱼精蛋白DNA阵列测得的力显示出与多价离子沉淀的DNA非常相似的特征。此外，我们还表明，重建的鲑鱼精蛋白DNA阵列和天然鲑鱼核给出了相同的渗透应激力曲线。我们假设雄性不育症因精素缺乏症和修饰中的误差是由于精子核中DNA的堆积而造成的，而不是最佳的，因此对诱变剂和氧化物种的可及性更大。 与较小的多价离子或精素凝结后，所得的压实结构的DNA双螺旋的平衡分离明确，取决于冷凝离子的身份，为0.7 1.5 nm。螺旋的有限分离表明，短距离排斥力的微妙平衡，吸引力更长。仍在争论着作用于紧凑多价离子组件的力的物理起源。我们曾根据我们以前对带电分子和未充电分子之间的力量进行广泛测量的水结构或水合力。 DNA组装的大多数理论都包含一种在一个螺旋上具有某种形式的正电荷相关性，并在另一种螺旋磷酸盐的另一个螺旋中，以说明吸引力。 使用渗透应力和单分子镊子实验，我们先前已经表征了两个组分力的距离依赖性。吸引力随DNA-DNA间距而变化为0.4 0.5 nm衰减长度指数；而排斥力为0.2 0.25 nm的衰减长度指数。衰减长度有两个差异的因素表明，带电组的直接相互作用在螺旋螺旋中的直接相互作用引起。而排斥力是图像充电或其水合当量的力。使用此约束，我们可以有效地将对力距离曲线的吸引力和排斥性贡献分开。 使用合成肽，我们能够阐明DNA精蛋白压实的几种特征。所有脊椎动物的精氨酸几乎都偏爱精氨酸，而不是赖氨酸。我们发现，与相同长度的赖氨酸肽相比，精氨酸肽可以将DNA组装到更紧密的间距上。基于精子核中赖氨酸的精蛋白的包装螺旋将使DNA更容易受到损害。我们假设两个氨基酸之间的结合几何形状差异会影响螺旋螺旋的电荷相关性。将中性氨基酸掺入精氨酸肽中会增加短量排斥力的振幅，同时仅略微影响吸引力。我们假设这些中性氨基酸通过从DNA凹槽中取代其他水，从而增加了图像电荷，例如排斥力。可以从精氨酸的数量和中性氨基酸的比例来定量预测鲑鱼精蛋白DNA复合物的排斥力和吸引力的振幅。将一个带负电荷的天冬氨酸或磷酸化丝氨酸掺入六边形精氨酸肽中会急剧增加短态排斥的幅度，这相当于掺入8种中性氨基酸。范围较长的吸引力适中减少，但与净+5电荷一致。这可能对精子发生具有生物学意义。精蛋白替换组蛋白的替代以几个步骤发生。第一个乙酰化组蛋白被过渡蛋白取代。丝氨酸磷酸化的精蛋白比取代过渡蛋白。只有在去除磷酸基团后，DNA才能紧密堆积。已经提出了不完全的去磷酸化原因导致DNA损伤增加和男性不育症。这里的结果表明，磷酸化对削弱螺旋之间的净吸引力的影响要大得多，而不是简单地减少精蛋白电荷所表明的影响。 我们在蔗糖梯度上有分级分离的鲑鱼精子核，并测量了DNA螺旋之间的X射线间距。沉积核的特征更慢，其特征是螺旋之间的平均间距更大，而与密度较差的分数相比。对散射峰的仔细分析表明，平均间距的增加是从正常距离到较大距离的间距离扩散增加的结果，而不是散射峰的简单偏移，该散射峰恒定宽度向更大的间距。 哺乳动物和piscine精蛋白之间存在几种差异。许多哺乳动物有两个精蛋白，P1和P2，两者都比Piscine精神病长。 50-60％的哺乳动物精蛋白的平均分数精氨酸明显小于鱼类，65-70％。根据我们的结果，预计中性氨基酸含量的增加将减少净吸引力，从而显着增加哺乳动物精子中螺旋之间的平衡间距。由于中性氨基酸的比例增加，DNA的宽松包装会增加诱变剂的可透性和反应性氧化物种对DNA的可及性，从而增加损伤。这可能解释了哺乳动物和piscine蛋白质DNA包装之间的另一个区别。哺乳动物精子中存在精蛋白之间的大量二硫键。另一方面，大多数piscine精蛋白没有半胱氨酸。紧密的DNA包装可能需要哺乳动物中的二硫键，从而克服了中性氨基酸的增加。 我们现在正在研究牛精子中的DNA包装。公牛只有P1精蛋白，代表了鱼类和人类精子之间复杂性的中间体。具有完整二硫键的牛精子核的渗透应激力曲线与鲑鱼精子核的渗透应力曲线非常相似。用DTT的二硫键还原会导致核的肿胀（体积至少10倍）和X射线顺序的完全损失。二硫键对于DNA的致密堆积绝对必要。减少牛核的渗透应激力曲线在较高的压力下逐渐与未修饰的牛精子核曲线合并。如果将DTT从极大肿胀的核中取出，则二硫键不会改革，但是如果去除DTT并用渗透应激压实核，则会改革。 未来的工作包括分离牛精蛋白，用于重新研究和密度分级和X射线分析以及牛核的X射线分析，并通过质谱法测定了8-氧气的8-氧气，作为氧化DNA损伤的指标。