Single Molecule Studies of Nucleic Acids Remodeling

项目摘要 通过Watson-Crick底线形成的双链DNA需要分离成单链 基因组复制或修复发生。同样，双链RNA经常被分离成单链 转录、剪接、核糖体生物发生和翻译过程中的链。这样的核酸解体 不应该不加区别地发生。使用先进的单分子测量技术，我们发现 通过构象开关调节解旋酶活性的新机制。这导致了 一种超螺旋酶的发展，它可以连续地解开数千个碱基对，即使在对抗强链的情况下也是如此 相反的力量。 核酸解离的一个镜像过程是两个核酸序列的蛋白质相关的退火法。 例如，Rec/RAD51介导的DNA重组，基于非编码RNA的基因调控和 基于CRISPR的DNA降解。所有这些过程都依赖于高于阈值的基本交互作用 表示特异性的BP数。如何快速、准确地识别目标DNA或RNA 大量过剩的其他序列的存在是一个悬而未决的问题。 蛋白质在解旋或解离方向上的“核酸重塑” 退火是这个项目的主旋律。这里的前提是基准线和基准线之间的平衡 在细胞内这些蛋白质的正常功能中，破坏和形成是至关重要的，如果平衡不是 如果维护得当，它会导致不当监管和疾病状态。要解决的关键问题是：(1)什么 不同的解旋酶构象在体内起作用吗？(2)我们能检测到所有的反应中间产物吗 解旋酶功能？(3)我们能用来模拟共转录的RNA折叠和核糖体组装吗？ 超螺旋酶？(4)碱基相互作用如何通过以下方式控制复制分叉的逆转和恢复 退火解旋酶？(5)碱基相互作用如何决定基于sRNA的基因的体内动力学 调控？(6)异源双链延伸和逆转之间的平衡如何控制CRISPR- Cas9/Cpf1靶向验证和切割激活？(7)异源双链之间的平衡如何 延伸和逆转控制Cas3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?