Ding Yue, Manzo Carlo, Fulcrand Geraldine, Leng Fenfei, Dunlap David, Finzi Laura
Departments of Physics and.
Department of Chemistry and Biochemistry, Florida International University, Miami, FL 33199.
Proc Natl Acad Sci U S A. 2014 Oct 28;111(43):15402-7. doi: 10.1073/pnas.1320644111. Epub 2014 Oct 15.
Topoisomerases, polymerases, and the chirality introduced by the binding of histones or nucleoid-associated proteins affect DNA supercoiling in vivo. However, supercoiling is not just a by-product of DNA metabolism. Supercoiling is an indicator of cell health, it modifies the accessibility of chromatin, and coordinates the transcription of genes. This suggests that regulatory, protein-mediated loops in DNA may sense supercoiling of the genome in which they are embedded. The λ repressor (CI) maintains the quiescent (lysogenic) transcriptome of bacteriophage λ in infected Escherichia coli. CI-mediated looping prevents overexpression of the repressor protein to preserve sensitivity to conditions that trigger virulence (lysis). Experiments were performed to assess how well the CI-mediated DNA loop traps superhelicity and determine whether supercoiling enhances CI-mediated DNA looping. CI oligomers partitioned plasmids into topological domains and prevented the passage of supercoiling between them. Furthermore, in single DNA molecules stretched and twisted with magnetic tweezers, levels of superhelical density confined in CI-mediated DNA loops ranged from -15% or +11%. Finally, in DNA under tensions that may occur in vivo, supercoiling lowered the free energy of loop formation and was essential for DNA looping. Supercoiling-enhanced looping can influence the maintenance of lysogeny in the λ repressor system; it can encode sensitivity to the energy level of the cell and creates independent topological domains of distinct superhelical density.
拓扑异构酶、聚合酶以及由组蛋白或类核相关蛋白结合引入的手性会影响体内的DNA超螺旋。然而，超螺旋不仅仅是DNA代谢的副产物。超螺旋是细胞健康的一个指标，它会改变染色质的可及性，并协调基因的转录。这表明DNA中由蛋白质介导的调控环可能会感知其所嵌入基因组的超螺旋状态。λ阻遏蛋白（CI）在受感染的大肠杆菌中维持噬菌体λ的静止（溶原）转录组。CI介导的环化可防止阻遏蛋白的过度表达，以保持对触发毒性（裂解）条件的敏感性。我们进行了实验，以评估CI介导的DNA环捕获超螺旋的能力，并确定超螺旋是否增强CI介导的DNA环化。CI寡聚体将质粒分隔成拓扑结构域，并阻止超螺旋在它们之间传递。此外，在用磁镊拉伸和扭转的单个DNA分子中，CI介导的DNA环内的超螺旋密度水平范围为-15%或+ 11%。最后，在体内可能出现的张力下的DNA中，超螺旋降低了环形成的自由能，并且对于DNA环化至关重要。超螺旋增强的环化可以影响λ阻遏蛋白系统中溶原状态的维持；它可以编码对细胞能量水平的敏感性，并创建具有不同超螺旋密度的独立拓扑结构域。
