Cotranscriptional R-loop formation by Mfd involves topological partitioning of DNA.
Cotranscriptional R-loop formation by Mfd involves topological partitioning of DNA.
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DOI：
10.1073/pnas.2019630118
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发表时间：
2021-04-13
影响因子：
11.1
通讯作者：
Strick TR
中科院分区：
文献类型：
--
作者：
Portman JR;Brouwer GM;Bollins J;Savery NJ;Strick TR
r环结构对基因组完整性构成威胁，因为它们的形成可能导致突变。突变驱动细菌的抗生素耐药性和人类癌症的化疗耐药性；因此，了解r环是如何形成的是很重要的。通过结合体外单分子实验和体内诱变实验，我们展示了Mfd（一种以其在DNA修复中的作用而闻名的细菌蛋白）如何与RNA聚合酶相互作用形成r环。这种相互作用在DNA中产生一个拓扑结构域，该结构域极易形成r环。观察到的机制依赖于许多其他蛋白质共有的Mfd特性，这暗示这是r环形成的潜在通用模型。r环是RNA侵入双工DNA与其模板序列配对时形成的核酸杂交体。尽管它们与越来越多的基因调控过程有关，但它们的机制起源仍不清楚。我们在此报告了在单分子分辨率下实时观察到的共转录r环形成，并提出了其形成的机制。我们发现细菌Mfd蛋白可以同时与伸长RNA聚合酶和上游DNA相互作用，将两者捆绑在一起，并将DNA划分为不同的超螺旋结构域。在Mfd和RNA聚合酶之间形成一个高度负的超卷曲结构域，在RNA聚合酶的前面和Mfd的后面出现补偿性的正超卷曲结构域。新生RNA侵入负超旋结构域，形成一个稳定的r环，可以驱动突变。从理论上讲，这种机制使任何同时结合主动易位RNA聚合酶和上游DNA的蛋白质都能刺激r环的形成。
R-loop structures pose a threat to genomic integrity because their formation can lead to mutagenesis. Mutagenesis drives antibiotic resistance in bacteria and chemotherapy resistance in human cancers; therefore, it is important to understand how R-loops form. Using a combination of in vitro single-molecule experimentation and in vivo mutagenesis assays, we have shown how Mfd, a bacterial protein known for its role in DNA repair, can interact with RNA polymerase to form R-loops. This interaction generates a topological domain in DNA that is highly prone to R-loop formation. The observed mechanism relies on properties of Mfd that are shared by many other proteins, hinting that this is a potentially universal model for R-loop formation. R-loops are nucleic acid hybrids which form when an RNA invades duplex DNA to pair with its template sequence. Although they are implicated in a growing number of gene regulatory processes, their mechanistic origins remain unclear. We here report real-time observations of cotranscriptional R-loop formation at single-molecule resolution and propose a mechanism for their formation. We show that the bacterial Mfd protein can simultaneously interact with both elongating RNA polymerase and upstream DNA, tethering the two together and partitioning the DNA into distinct supercoiled domains. A highly negatively supercoiled domain forms in between Mfd and RNA polymerase, and compensatory positive supercoiling appears in front of the RNA polymerase and behind Mfd. The nascent RNA invades the negatively supercoiled domain and forms a stable R-loop that can drive mutagenesis. This mechanism theoretically enables any protein that simultaneously binds an actively translocating RNA polymerase and upstream DNA to stimulate R-loop formation.
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DOI：
10.1073/pnas.1807031116
发表时间：
2019-02-12
影响因子：
11.1
作者：
Ma, Jie;Tan, Chuang;Wang, Michelle D.
通讯作者：
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影响因子：
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作者：
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通讯作者：
HANAWALT, PC
影响因子：
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通讯作者：
SANCAR, A
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10.1073/pnas.1416651112
发表时间：
2015-03-10
影响因子：
11.1
作者：
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Merrikh, Houra
DOI：
10.1111/j.1432-1033.1970.tb00287.x
发表时间：
1970-01-01
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
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作者：
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通讯作者：
STEIN, H