Collaborative Research: Topological Characterization of DNA Organization in Bacteriophages

大分子自组装过程是发生在每个生物体的复杂相互作用网络中的关键角色。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病毒的形成。在双链DNA （dsDNA）病毒中，这一途径的两个关键和相互关联的方面仍然是未知的，即病毒内部的包装反应和随后的DNA基因组折叠。该项目的目的是取得进展，以更好地了解这种DNA的包装和折叠在压缩条件下一般。该项目的长期目标是提供dsDNA病毒中DNA包装和折叠过程的详细定量描述。提案中有两个具体目标。第一个是重新审视和改进目前噬菌体中DNA折叠的模型。这些DNA折叠模型将使用计算机模拟进行打结测试。pi将决定它们产生的结分布与在噬菌体P4中实验观察到的结分布的匹配程度。此信息将用于指导拒绝或修改和改进模型。这些改进模型的物理参数，如链的柔韧性将被估计。所得到的模型将通过它们是否产生可以与冷冻电镜数据进行比较的DNA密度图的标准进行验证。第二个目标是利用拓扑和随机过程重建噬菌体P4内部的DNA折叠。从实验中观察到的DNA结数据暗示了噬菌体中DNA包装的一定程度的随机性。从一个完全随机的模型开始，使用P4中的结分布作为指导，pi将开发一个模型来解释实验观察到的结。该模型将与第一个目标中获得的模型以及来自CryoEM数据的DNA密度图进行比较。双链DNA （dsDNA）病毒引起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，就需要更好地了解感染性病毒产生和复制的自组装途径。在实验室获得的结果只提供了部分信息。部分原因是由于DNA在包装和复制过程中受到极端程度的凝结，DNA的行为很难通过实验来检测。此外，虽然提出了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将利用一种叫做“结理论”的工具，建立数学模型，开发和测试一种更精细的dsDNA病毒DNA包装和折叠理论。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，可以更充分地说明实验数据。这项工作将促进对病毒DNA折叠和包装的认识，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，这项工作将推进约束下随机行走的数学和计算理论。由于随机游走理论在其他科学研究中是一个重要且常用的工具，用于处理随机串状物体（如长聚合物链），因此这项工作也可以潜在地造福于其他科学领域。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.