Detection of cellular G-quadruplex by using a loop structure as a structural determinant
Detection of cellular G-quadruplex by using a loop structure as a structural determinant
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DOI：
10.1016/j.bbrc.2020.05.191
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发表时间：
2020-10-08
影响因子：
3.1
通讯作者：
Fukatsu, Rino
中科院分区：
文献类型：
--
作者：
Masai, Hisao;Kanoh, Yutaka;Fukatsu, Rino
现在已知G-quadrupex在各种生物反应中起着至关重要的作用。然而，由于缺乏通用和无偏见的方法，其在细胞中存在的直接证据有限。我们使用Rif 1结合位点的裂变酵母基因组，已被证明采用G4结构，作为一个模型，以证明Rif 1 BS确实采取G4结构在细胞中。我们利用G4结构中存在的单链环。Rif 1BS的独特之处在于它们含有异常长的环序列，我们用18 bp的I-SceI限制性位点取代了它们。我们表明，在体外，I-SceI在环不裂解时，G4形成双链Rif 1BS DNA，但裂解时，G4不形成由于在G-tracts的突变。这在热诱导和转录诱导的G4结构中都观察到，并为这一过程提供了证据。我们应用这种策略检测的G4结构在相同的Rif 1BS在裂殖酵母细胞。我们提出的证据表明，在体内切割的I-SceI可以是一个措施，在细胞中的靶序列的G4的存在。这里描述的方法提供了一个平台策略，用于细胞G4及其动态形成和破坏的全基因组分析。(C)2020爱思唯尔公司All rights reserved.
G-quadrupex is now known to play crucial roles in various biological reactions. However, direct evidence for its presence in cells has been limited, due to the lack of versatile and non-biased methodology. We use Rif1 binding sites on the fission yeast genome, which has been shown to adopt G4 structures, as a model to prove that Rif1 BS indeed adopt G4 structure in cells. We take advantage of the presence of a single-stranded loop in the G4 structure. Rif1BS is unique in that they contain unusually long loop sequences, and we replace them with a 18 bp I-SceI restriction site. We show in vitro that I-SceI in the loop is not cleaved when G4 is formed on duplex Rif1BS DNA, but is cleaved when G4 is not formed due to a mutation in the G-tracts. This is observed both heat-induced and transcription-induced G4 structure, and gives proof of evidence for this procedure. We apply this strategy for detection of a G4 structure at the same Rif1BS in fission yeast cells. We present evidence that in vivo cleavage of I-SceI can be a measure for the presence of G4 at the target sequence in cells as well. The method described here gives a platform strategy for genome-wide analyses of cellular G4 and their dynamic formation and disruption. (C) 2020 Elsevier Inc. All rights reserved.