Single Molecule Studies of Nucleic Acids Remodeling

项目摘要 通过沃森-克里克碱基配对形成的双链DNA需要被分离成单链， 基因组复制或修复。同样，双链RNA通常被分离成单链RNA。 在转录、剪接、核糖体生物合成和翻译过程中的单链。这样的核酸解旋 不应该不加区别地发生。利用先进的单分子测量技术， 通过构象转换调节解旋酶活性的新机制。这导致 开发一种超级解旋酶，即使对强的 反对力量。 核酸解旋的镜像过程是两个核酸链的蛋白质依赖性退火。 实例包括Rec/Rad 51介导的DNA重组、基于非编码RNA的基因调控和 CRISPR DNA降解所有这些过程都依赖于阈值以上的碱基配对相互作用 特异性的bp数。如何快速准确地鉴定靶DNA或RNA， 大量过量存在其它序列是一个未回答的问题。 由蛋白质在解旋方向或解旋方向介导的“核酸重塑” 退火是这个项目的首要主题。这里的前提是碱基对之间的平衡 打破和形成是至关重要的正常功能，这些蛋白质在细胞内，如果不平衡 如果保养不当，就会导致失调和疾病状态。要解决的关键问题是：（1）什么 是各种解旋酶构象在体内的作用？(2)我们能检测到所有的反应中间体， 解旋酶功能？(3)我们能模拟共转录RNA折叠和核糖体组装， 超级解旋酶(4)碱基配对相互作用如何控制复制叉逆转和恢复， 退火解旋酶？(5)碱基配对相互作用如何决定基于sRNA的基因的体内动力学 监管？(6)异源双链延伸和逆转之间的平衡如何控制CRISPR- Cas9/Cpf 1靶标验证和切割激活？(7)异源双链之间如何平衡 延伸和逆转控制Cas 3解旋酶-核酸酶募集？

Project Summary Double stranded DNA formed via Watson-Crick basepairing needs to be separated into single strands for genome duplication or repair to occur. Likewise, double stranded RNA are frequently separated into single strands during transcription, splicing, ribosome biogenesis and translation. Such nucleic acids unwinding should not occur indiscriminately. Using advanced single molecule measurement technologies we discovered a novel mechanism of regulating helicase activities through a conformational switch. This led to the development of a superhelicase that can unwind thousands of base pairs processively even against a strong opposing force. A mirror process to nucleic acids unwinding is protein-dependent annealing of two stands of nucleic acids. Examples include Rec/Rad51-mediated DNA recombination, non-coding RNA-based gene regulation and CRISPR-based DNA degradation. All of these processes rely on basepairing interactions above the threshold number of bp for specificity. How the target DNA or RNA can be both rapidly and accurately identified in the presence of other sequences in large excess is an unanswered question. `Nucleic acids remodeling' mediated by proteins either in the direction of unwinding or in the direction of annealing is the overarching theme of this project. The premise here is that a balance between the basepair breaking and formation is critical in normal functions of these proteins inside the cell and if the balance is not properly maintained, it leads to mis-regulation and diseased states. The key questions to address are: (1) What is the in vivo role of various helicase conformations? (2) Can we detect all reaction intermediates during helicase function? (3) Can we mimic co-transcriptional RNA folding and ribosome assembly using superhelicases? (4) How do basepairing interactions control replication fork reversal and restoration by annealing helicases? (5) How do basepairing interactions determine in vivo kinetics of sRNA-based gene regulation? (6) How does the balance between heteroduplex extension and reversal control CRISPR- Cas9/Cpf1 target verification and cleavage activation? (7) How does the balance between heteroduplex extension and reversal control Cas3 helicase-nuclease recruitment?