Assembly Forces And Recognition Reactions

装配力和识别反应

• 批准号: 8351169
• 负责人: Donald Rau
• 金额: $ 25.22万
• 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/8351169
• 关键词: Accounting; Agreement; Alanine; Amino Acids; Arginine; Bacteriophages; Binding; Biological; Cations; Cell Nucleus; Cell physiology; Cellular biology; Characteristics; Charge; Chromatin; Complex; Coupled; Coupling; Cysteine; DNA; DNA Damage; DNA Packaging; DNA Sequence; DNA delivery; Data; Defect; Dependence; Disease; Dissociation; Disulfides; Equilibrium; Fishes; Genetic; Genome; Glutamates; Goals; Histones; Homo; Hydration status; Image; Infertility; Ions; Isoleucine; Knowledge; Length; Link; Male Infertility; Mammals; Manganese; Measurement; Measures; Modeling; Modification; Mutagens; Neutral Amino Acids; Nuclear Proteins; Oligopeptides; Osmotic Pressure; Peptides; Phosphorylation; Physical condensation; Physics; Proline; Property; Protamines; Protein Binding; Protein Dephosphorylation; Proteins; Reaction; Salmon; Sequence-Specific DNA Binding Protein; Series; Serine; Solutions; Specificity; Spermatids; Spermatogenesis; Spermidine; Spermine; Stress; Structure; Techniques; Water; aqueous; base; density; design; expectation; hexaamminecobalt(II); inorganic phosphate; macromolecule; nucleic acid structure; physical property; reconstitution; research study; single molecule; sperm cell; structural biology; theories

During vertebrate spermatogenesis, chromatin is dramatically reorganized in developing spermatids via histone replacement with protamines to achieve an even more compacted state for efficient genetic delivery and DNA protection. Dense packaging of DNA in sperm nuclei is considered important to protect the DNA against damage by mutagens or oxidative species. Protamines are small, arginine-rich, nuclear proteins that condense the spermatid genome into a genetically inactive state. Salmon protamine is a 32 amino acid peptide with 21 arginines and 11 neutral amino acids (PR4S3RPVR5PRVSR6G2R4). 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 both reconstituted protamine DNA arrays and native salmon nuclei by the osmotic stress technique coupled with x-ray diffraction show very similar characteristics to DNA precipitated by multivalent ions. 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 7 - 15 , 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 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 4 - 5 decay length exponential; whereas the repulsive force is a 2 - 2.5 decay length exponential. The factor of two in decay lengths suggests that the attractive force results from a direct interaction of charged groups on apposing helices; whereas the repulsive force seems to be 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. The amplitudes of the two forces were extracted from the force curves for a set of homologous arginine peptides, R1 through R6 and poly-arginine. The magnitude of the attractive force depends on the arginine peptide charge. The repulsive force amplitude, in contrast, is nearly independent of arginine peptide length. The equilibrium spacing between DNA helices in reconstituted +21 charged salmon protamine DNA arrays was only equivalent to +5 charged R5. This is surprising since it is generally considered that cation charge is the dominating determinant of DNA compaction. The separation of the forces for salmon protamine-DNA assemblies indicated that the attractive force amplitude with protamine was very close to that expected for 21 arginines, but that the repulsive force had a larger amplitude than expected from the homo-arginine peptide series. This additional repulsion results in the observed lower packaging efficiency of +21 charged protamine compared to +6 hexa-arginine. Salmon protamine is long enough that the extra repulsion could be due to defects in its binding along the DNA helix. In order to determine if this extra repulsion is a general property of including neutral amino acids in arginine based peptides or if it is something peculiar to protamine, we investigated the effect on forces of inserting neutral amino acids into model hexa-arginine (R6) peptides. Neutral amino acids inserted into R6 significantly increase the amplitude of short ranged repulsive force. We observe a smaller decrease in the amplitude of the attraction. The effect on force amplitudes of incorporating increasing numbers of alanines into the middle of R6 is additive at least through four. There is relatively little difference in inserting alanine, serine, proline, or isoleucine. We find that the increase in the short ranged repulsive force for salmon protamine condensed DNA is in reasonablequantitative agreement with its neutral amino acid content. We also investigated the effect of incorporating a single negative charge, glutamate or phosphorylated serine, into R6. The net attraction between DNA helices is substantially weakened, much more than simply reducing the net charge by one. Indeed, the equilibrium spacing is significantly greater that seen with R3. The short-ranged repulsion is dramatically increased. The longer ranged attraction is moderately decreased, but is consistent with a net +5 charge. There are several differences between mammalian and piscine protamines. Most but not all 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. Not surprisingly, the incorporation of a negatively charged amino acid into R6 has a large effect on forces. 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 recently begun examination of mammalian sperm nuclei and are still optimizing techniques. Our preliminary data indicates that with intact disulfide bridges stallion sperm DNA is packaged as tightly as in salmon sperm. The spacing between helices in stallion sperm greatly increases when the disulfide bridges are reduced in agreement with our expectations. We expect to find a correlation between DNA packing densities and infertility.

在脊椎动物的精子发生过程中，染色质在通过食组蛋白替换蛋白质中大量重新组织，以实现更紧凑的状态，以实现有效的遗传递送和DNA保护。精子核中DNA的致密包装对于保护DNA免受诱变剂或氧化物种的损害很重要。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。鲑鱼精素是一种32个氨基酸肽，具有21氨基氨酸和11个中性氨基酸（PR4S3RPVR5PRVSR6G2R4）。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，通过渗透应激技术与X射线衍射相结合的渗透应力技术对重构的精蛋白DNA阵列和天然鲑鱼核测量的力显示与多价离子沉淀的DNA非常相似。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，根据冷凝离子的身份，所得的压实结构的DNA双螺旋的平衡分离明确定义为7-15。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。我们根据我们先前对带电分子和未充电分子之间的力量进行了广泛的测量，主张水结构或水合力。 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。吸引力随着DNA -DNA间距为4-5衰减长度指数而变化。而排斥力是2-2.5衰减长度指数。衰减长度的两个因子表明，引起的吸引力是由带电组的直接相互作用引起的。而排斥力似乎是图像充电或其水合等效力。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。从力曲线中提取了两种力的幅度，以作为一组同源精氨酸肽R1至R6和多精氨酸。吸引力的大小取决于精氨酸肽电荷。相反，排斥力振幅几乎与精氨酸肽长度无关。重构+21带电的鲑鱼精蛋白DNA阵列中DNA螺旋之间的平衡间距仅等于+5带电的R5。这是令人惊讶的，因为通常认为阳离子电荷是DNA压实的主要决定因素。鲑鱼精蛋白-DNA组件的力的分离表明，与精氨酸的吸引力振幅非常接近21精氨酸的预期，但是排斥力的振幅比同型精氨酸肽系列的预期更大。与+6 Hexa-精氨酸相比，这一额外的排斥导致观察到的+21带电精蛋白的包装效率较低。鲑鱼精神足够长，以至于额外的排斥可能是由于其沿着DNA螺旋的结合中的缺陷所致。为了确定这种额外的排斥是在基于精氨酸的肽中包含中性氨基酸的一般特性，还是精神蛋白特有的东西，我们研究了对将中性氨基酸插入模型Hexa-精氨酸（R6）肽的作用的影响。 。 插入R6中的中性氨基酸显着增加了远程排斥力的幅度。我们观察到吸引力的幅度较小。至少至少四个，对将增加的丙氨酸数量掺入R6中部的力振幅的影响是加性的。插入丙氨酸，丝氨酸，脯氨酸或异亮氨酸的差异相对较小。我们发现，鲑鱼精素凝结DNA的短距离排斥力的增加与中性氨基酸含量合理吻合。 我们还研究了将单个负电荷（谷氨酸或磷酸化丝氨酸）纳入R6的效果。 DNA螺旋之间的净吸引力大大削弱了，不仅仅是将净电荷减少一个。实际上，平衡间距明显大于R3所见。短期排斥力大大增加。范围较长的吸引力适中减少，但与净+5电荷一致。 哺乳动物和piscine精蛋白之间存在几种差异。大多数但并非所有哺乳动物都有两个精蛋白，P1和P2，两者都比Piscine精蛋白更长。 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。 毫不奇怪，将带负电荷的氨基酸掺入R6对力的影响很大。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。 我们最近开始研究哺乳动物的精子核，并且仍在优化技术。我们的初步数据表明，使用完整的二硫键桥，种马的精子DNA与鲑鱼精子一样紧密。当二硫键与我们的期望一致时，种马精子中的螺旋螺旋架之间的间距大大增加。我们期望在DNA填料密度和不育之间找到相关性。