Periodic DNA patrolling underlies diverse functions of Pif1 on R-loops and G-rich DNA.
Periodic DNA patrolling underlies diverse functions of Pif1 on R-loops and G-rich DNA.
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作者：
Zhou R;Zhang J;Bochman ML;Zakian VA;Ha T
pif 1家族解旋酶从细菌到人类都是保守的。在这里，我们报告了一种新的DNA巡逻活动，这可能是Pif 1的多种功能的基础：Pif 1单体优先将自身锚定在3′-尾DNA连接处，并以一个核苷酸的步长周期性地缠绕3′-尾，挤出一个环。这种周期性的巡逻活动用于在每次相遇时展开分子内的G-四链体（G4）结构，并且足以展开RNA-DNA异源双链体而不是双链体DNA。而不是离开后G4解旋，让它重新折叠，或超越解开双链体DNA，Pif 1反复解旋G4 DNA，保持它展开。Pif 1诱导的G4解折叠发生在三个离散的步骤中，一次一条链，并且足以克服G4稳定药物。周期性的巡逻活动可以保持Pif 1在其体内作用位点，以取代端粒酶，解析R环，并在复制，重组和修复过程中保持G4未折叠。解旋酶是一种酶，以其分离构成著名的双螺旋结构的两条DNA链的能力而闻名。http://dx.doi.org/10.7554/eLife.02190.001细胞内的许多重要过程，包括基因作为蛋白质的表达，以及细胞分裂前DNA的复制，都依赖于DNA分子以这种方式被分离。然而，这些酶可以发挥许多其他作用，帮助维持细胞DNA的完整性。遗传密码是使用四个DNA碱基（称为A、C、G和T）编写的，如果一段DNA包含大量G碱基，那么其中一条链可以自身环回三次，形成一种称为“G”的结构-四倍体”。这些结构可以阻止基因的表达，减缓DNA的复制。然而，一种称为Pif 1的解旋酶可以解开G-四链体，使这些活动继续进行。这种解旋酶存在于从细菌到人类的许多生物体中，并为细胞执行多种功能。然而，这些活动背后的确切机制尚不清楚。现在，Zhou等人使用生物物理技术揭示了单个Pif 1蛋白质与DNA分子一端的单链突出端结合。Pif 1还与DNA中的叉结合，在那里双螺旋分离成两条单链。一旦Pif 1与DNA结合，它就会一次一个碱基地“卷”入突出端或单链。这种活动可以解开一个G-四链体，单个Pif 1蛋白将巡逻DNA，以保持这种结构解开，而不会解开双螺旋本身。分离两条DNA链实际上需要多个Pif 1蛋白质连接并一起工作。当它巡逻时，Pif 1也会从DNA中取代其他蛋白质，并去除DNA中不寻常的、可能有害的结构（例如取代DNA双螺旋链之一的RNA分子）。下一个挑战将是解决仍然没有答案的重要问题，包括：Pif 1如何识别DNA结构并改变其活性;以及它如何与靶向相同结构的其他蛋白质协调。DOI：http://dx.doi.org/10.7554/eLife.02190.002网站
Pif1 family helicases are conserved from bacteria to humans. Here, we report a novel DNA patrolling activity which may underlie Pif1’s diverse functions: a Pif1 monomer preferentially anchors itself to a 3′-tailed DNA junction and periodically reel in the 3′ tail with a step size of one nucleotide, extruding a loop. This periodic patrolling activity is used to unfold an intramolecular G-quadruplex (G4) structure on every encounter, and is sufficient to unwind RNA-DNA heteroduplex but not duplex DNA. Instead of leaving after G4 unwinding, allowing it to refold, or going beyond to unwind duplex DNA, Pif1 repeatedly unwinds G4 DNA, keeping it unfolded. Pif1-induced unfolding of G4 occurs in three discrete steps, one strand at a time, and is powerful enough to overcome G4-stabilizing drugs. The periodic patrolling activity may keep Pif1 at its site of in vivo action in displacing telomerase, resolving R-loops, and keeping G4 unfolded during replication, recombination and repair. DOI: http://dx.doi.org/10.7554/eLife.02190.001 Helicases are enzymes that are best known for their ability to separate the two strands of DNA that make up the famous double-helix structure. Many important processes within cells—including the expression of genes as proteins, and the replication of DNA before cell division—rely on DNA molecules being separated in this way. However, these enzymes can perform many other roles that help maintain the integrity of a cell’s DNA. The genetic code is written using four DNA bases—called A, C, G and T—and if a stretch of DNA contains lots of G bases, then one of the strands can loop back upon itself three times to form a structure known as a ‘G-quadruplex’. These structures can prevent the expression of genes, and slow the replication of DNA. However, a helicase called Pif1 can unwind G-quadruplexes to allow these activities to continue. This helicase is found in many organisms, from bacteria to humans, and carries out multiple functions for a cell. However, the exact mechanisms underlying these activities are unknown. Now, Zhou et al. have used biophysical techniques to reveal that individual Pif1 proteins bind to single-stranded overhangs at one end of a DNA molecule. Pif1 also binds to forks in DNA where the double helix separates into two single strands. And once Pif1 has bound to the DNA, it works to ‘reel in’ the overhang or a single strand, one base at a time. This activity can unwind a G-quadruplex, and individual Pif1 proteins will patrol DNA to keep this structures unwound without unraveling the double helix itself. Separating the two strands of DNA actually needs multiple Pif1 proteins to join and work together. As it patrols, Pif1 also displaces other proteins from DNA and removes unusual, and potentially harmful, structures in DNA (such as RNA molecules that have displaced one of the strands of DNA double helix). The next challenge will be to address important questions that remain unanswered including: how does Pif1 recognize DNA structures and change its activity; and how does it coordinate with other proteins that target the same structures? DOI: http://dx.doi.org/10.7554/eLife.02190.002