Collaborative Research: Topological Characterization of DNA Organizations in Bacteriophage Capsids

在每个生物体中发生的复杂的相互作用网络中，大分子自组装过程是关键。在病毒中，病毒蛋白和包装基因组的适当自组装决定了感染性病毒的形成。这一途径的两个关键和相互关联的方面在双链DNA(DsDNA)病毒中仍然是未知的，这两个方面是病毒内DNA基因组的包装反应和随后的折叠。这个项目的目的是为了更好地理解这种DNA在浓缩条件下的包装和折叠。该项目的长期目标是对dsDNA病毒的DNA包装和折叠过程提供详细的定量描述。该提案有两个具体目标。第一个是回顾和改进目前噬菌体中DNA折叠的模型。这些DNA折叠模型将通过计算机模拟进行打结测试。PI将确定它们产生的结的分布与在噬菌体P4中实验观察到的结的分布有多接近。这些信息将被用来指导拒绝或修改和改进模型。将对这些改进模型的链灵活性等物理参数进行估计。最终得到的模型将根据标准进行验证，以确定它们是否产生可与CryoEM数据进行比较的DNA密度图。第二个目标是利用拓扑学和随机过程重建噬菌体P4内部的DNA折叠。从实验上观察到的DNA结的数据表明，噬菌体中的DNA包装具有一定程度的随机性。从一个完全随机的模型开始，使用P4中的节点分布作为指导，PI将开发一个模型来解释通过实验观察到的节点。这个模型将与第一个目标中获得的模型进行比较，也将与来自CryoEM数据的DNA密度图进行比较。双链DNA(DsDNA)病毒会导致从普通感冒到某些癌症的许多人类疾病。如果要开发药物和疫苗来中和这些病毒的影响，就需要更好地了解产生和复制感染性病毒的自组装途径。在实验室中获得的结果只提供了部分信息。部分原因是因为DNA在包装和复制过程中受到极端程度的凝聚，DNA的行为很难通过实验检测到。此外，尽管已经提出了DNA包装过程的各种模型，但这些模型都不能充分解释已获得的实验数据。PI将使用数学建模，使用一种名为纽结理论的工具，来开发和测试dsDNA病毒中DNA包装和折叠的更精细理论。基于现有模型的计算机模拟将与在噬菌体P4上获得的实验数据进行测试，以确定模型的哪些特征最能预测实验结果。这项工作的目的是产生一个修正的模型，可以更全面地解释实验数据。这项工作将促进对病毒中DNA折叠和包装的了解，并将对染色体结构和动力学的研究做出重要贡献，从而更好地理解复制、转录、分离和修复的生物学过程。此外，这项工作还将推进禁闭条件下随机游动的数学和计算理论。由于随机游走理论是处理随机弦状物体(如长聚合物链)的其他科学研究中重要且常用的工具，这项工作也可能有益于其他科学领域。通过该项目开发的研究工具将免费提供给科学界。

Macromolecular self-assembly processes are key players in the complex network of interactions that take place in every organism. In viruses proper self-assembly of the viral proteins and of the packaged genome determine the formation of an infective virus. Two critical and interrelated aspects of this pathway that remain mostly unknown in double-stranded DNA (dsDNA) viruses are the packing reaction and the subsequent folding of the DNA genome inside the virus. The aim of this project is to make progress towards a better understanding of such DNA packing and folding under a condensed condition in general. The long-term goal of this project is to provide a detailed quantitative description of the DNA packing and folding processes in dsDNA viruses. There are two specific objectives in the proposal. The first one is to revisit and improve current models of DNA folding in bacteriophages. These models of DNA folding will be tested for knotting using computer simulations. The PIs will determine how closely the knot distributions they generate match the distributions of knots observed experimentally in bacteriophage P4. This information will be used to guide the rejection or modification and improvement of the models. Physical parameters such as the chain flexibility of these improved models will be estimated. Resulting models will be validated by the criterion whether they produce DNA density maps that can be compared to cryoEM data. The second objective is to reconstruct the DNA folding inside bacteriophage P4 using topology and stochastic processes. Data from DNA knots observed experimentally implicates a certain degree of randomness in the DNA packing in bacteriophages. Starting from a completely random model and using the knot distribution from P4 as a guide, the PIs will develop a model that accounts for the knots observed experimentally. This model will be compared against models obtained in the first objective and also against DNA density maps from CryoEM data.Double-stranded DNA (dsDNA) viruses cause a number of human diseases ranging from the common cold to certain cancers. If drugs and vaccines are to be developed to neutralize the effects of these viruses, a better understanding of the self-assembly pathway through which infective viruses are produced and replicated is needed. Results obtained in the laboratory have yielded only partial information. Part of the reason is that because the extreme levels of condensation to which DNA is subjected during packing and replication, the behavior of the DNA is difficult to detect experimentally. Furthermore, though various models of the process of DNA packing have been proposed, none of these adequately accounts for the experimental data that have been obtained. The PIs will employ mathematical modeling, using a tool called knot theory, to develop and test a more refined theory of DNA packing and folding in dsDNA viruses. Computer simulations based on existing models will be tested against experimental data obtained on bacteriophage P4 to determine which features of the models best predict the experimental results. The aim of this work is to generate a revised model which can more fully account for the experimental data. This work will advance knowledge of DNA folding and packing in viruses and will make an important contribution to the study of chromosome structure and dynamics, leading to a better understanding of biological processes of replication, transcription, segregation and repair. Additionally, this work will advance the mathematical and computational theory of random walks under confinement. Since random walk theory is an important and commonly used tool in other scientific studies dealing with random string like objects (such as long polymer chains), this work can potentially benefit other fields of science as well. Research tools developed through the project will be made freely available to the scientific community.