Detection of cellular G-quadruplex by using a loop structure as a structural determinant
| Autor: | Yutaka Kanoh, Hisao Masai, Naoko Kakusho, Rino Fukatsu |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Direct evidence Telomere-Binding Proteins Biophysics Nucleic Acid Denaturation Cleavage (embryo) G-quadruplex Biochemistry 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Schizosaccharomyces Binding site Molecular Biology Binding Sites Chemistry Cell Biology Yeast G-Quadruplexes Restriction site 030104 developmental biology 030220 oncology & carcinogenesis Restriction digest Schizosaccharomyces pombe Proteins Genome Fungal DNA |
| Zdroj: | Biochemical and Biophysical Research Communications. 531:75-83 |
| ISSN: | 0006-291X |
| DOI: | 10.1016/j.bbrc.2020.05.191 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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