Journal: Nucleic Acids Research
Article Title: RPA hyperphosphorylation hinders the resolution of R-loops and G-quadruplex-associated R-loops during RAS-driven senescence
doi: 10.1093/nar/gkag331
Figure Lengend Snippet: G-loops exhibit resistance to RNase H1-mediated cleavage, suggesting a structural configuration that impairs enzymatic accessibility. ( A ) Motif discovery analysis reporting the three most frequent motifs found enriched in G4 ChIP-seq in BJ + RAS cells and co-localizing with R-loops within 3 kb. Each motif was analyzed using G4Hunter to identify the optimal sequence containing a putative QPS. The G4Hunter score is reported along with the predicted tetrads. ( B ) Plot reporting the distances in BJ + RAS of the G4s (corresponding to the three motifs found in Fig. ) with respect to the closest R-loop, divided according to their co-occurrence on either the template or non-template strand. ( C ) Heatmap showing the signal intensity of G4 structures and DRIP-seq in BJ + RAS cells, relative to the indicated genomic coordinates and the presence of QPSs marked by red arrows. In the case of the KRAS locus, these correspond to the well-characterized G4 regions referred to as ‘near’, ‘mid’, and ‘far’. ( D ) Metaplot of DRIP-seq and G4, RNA PolII Ser5 and BRCA1 ChIP-seq signals in BJ + RAS and BJ + RAS/HDAC4 cells within 6kb of “G-loop like” structures (R-loops co-occurring with G4 in non-template strand) identified in BJ + RAS cells. ( E ) The in vitro assay was performed by incubating equimolar amounts (800 pmol) of Cy5.5-labeled R-loops, G-loops containing the G4 motif “A” (ref. Fig. ) or DNA:RNA duplex (Cy5.5 labeling at 5′ of RNA) with increasing amounts of RNase H1. Native gel electrophoresis was performed to separate the various species of nucleic acids, which were schematized on the side. The fluorescence of the Cy5.5 labeled RNAs was acquired with the fluorescence reader and then the gel was stained with EtBr to detect the DNA duplexes with the transilluminator. ( F ) Histogram representing the ribonuclease efficiency of RNase H1 against synthetic R-loop and G-loop substrates (increasing amounts: 80, 160, and 400 pmol). Lanes corresponding to gel electrophoresis (in E) are indicated. Structures containing G4 in displaced DNA strand are in black (lanes 8–12). ( G ) Histogram representing the increase in fluorescence of Thioflavin T in the presence of substrates containing G4. Fluorescence emission was collected at 485 nm after excitation at 425 nm and expressed as increase of fluorescence quantum yield at 485 nm in the presence of G4-forming sequences in displaced DNA strand (lanes 8–12, increasing amounts: 80, 160, and 400 pmol). ( H ) Dot blot analysis using BG4 antibody to detect G4. Increasing amount of the indicated synthetic DNA:RNA structures (DNA:RNA heteroduplex not containing G4, R-loop not containing G4, G-loop with motif A, G-loop with 32R G4) were spotted on nitrocellulose film. RNA was Cy5.5 labeled at 5′. Cy5.5 fluorescence was used as loading control. ( I ) Fluorescence anisotropy binding assays showing the interaction between catalytically inactive RNase H1 (deadRNase H1) and the indicated nucleic acid substrates. Binding curves were fitted to determine the apparent dissociation constants ( K d ) for each substrate. In F and G, data are expressed as mean ± SD; n = 4. * P < 0.05, ** P < 0.01, *** P < 0.05. Pairwise t-test was applied to indicated comparisons.
Article Snippet: DNA SMART ChIP-Seq Kit (Takara) was used for library generation of samples subjected to Illumina sequencing.
Techniques: ChIP-sequencing, Sequencing, In Vitro, Labeling, Nucleic Acid Electrophoresis, Fluorescence, Staining, Dot Blot, Control, Binding Assay
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