Journal: Nucleic Acids Research

Article Title: Genome-wide CRISPR screens identify the EXO1-CAF-1 pathway suppressing R-loop-associated DNA damage

doi: 10.1093/nar/gkag226

Figure Lengend Snippet: EXO1 and CHAF1A are recruited to R-loops and synergistically suppress R-loop accumulation. ( A ) BrdU alkaline comet assay showing that CHAF1A depletion causes increased accumulation of replication-associated single stranded DNA lesions in both EXO1-knockout lines compared to WT HeLa cells. At least 100 nuclei were quantified for each condition. The median values are marked on the graph and listed at the top. Asterisks indicate statistical significance (Mann–Whitney, two-tailed). Schematic representations of the assay conditions are shown at the top. ( B, C ) S9.6 PLA experiments showing increased EXO1 ( B ) and CHAF1A ( C ) recruitment to R-loops in HeLa cells. At least 100 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance ( t -test, two-tailed, unpaired). ( D, E ) S9.6-dsDNA PLA experiments showing increased R-loops in HeLa cells with concomitant inactivation of EXO1 and CHAF1A. RNAseH1 overexpression suppresses the PLA signal, indicating that it derives from R-loops. At least 100 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance ( t -test, two-tailed, unpaired).

Article Snippet: Antibodies used were: S9.6 (Kerafast ENH001), double-stranded DNA (dsDNA; Novus NBP3-07302), γH2AX (Abcam ab2893), EXO1 (Novus NBP2-16391), and CHAF1A (Cell Signaling Technology 5480s).

Techniques: Alkaline Single Cell Gel Electrophoresis, Knock-Out, MANN-WHITNEY, Two Tailed Test, Over Expression

Journal: Nucleic Acids Research

Article Title: Genome-wide CRISPR screens identify the EXO1-CAF-1 pathway suppressing R-loop-associated DNA damage

doi: 10.1093/nar/gkag226

Figure Lengend Snippet: Concomitant depletion of EXO1 and CHAF1A causes the accumulation of R-loop-associated DNA damage. ( A ) S9.6-γH2AX PLA experiments showing increased R-loop-associated DNA damage in HeLa cells with concomitant inactivation of EXO1 and CHAF1A. RNAseH1 overexpression suppresses the PLA signal, indicating that it derives from R-loops. At least 100 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance ( t -test, two-tailed, unpaired). The siCHAF1A/siControl mean ratios are also presented. ( B–D ) γH2AX immunofluorescence showing that CHAF1A depletion causes increased DNA damage in both EXO1-knockout lines compared to WT HeLa cells, which is suppressed by RNaseH1 overexpression. Quantifications ( B, D ) and representative micrographs with scale bars representing 10 µm ( C ) are shown. At least 100 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance ( t -test, two-tailed, unpaired). ( E–G ) Neutral comet assays showing that CHAF1A depletion causes increased DSB formation in both EXO1-knockout lines compared to WT HeLa cells, which is suppressed by RNaseH1 overexpression. Quantifications ( E, G ) and representative micrographs with scale bars representing 10 µm ( F ) are shown. At least 100 comets were quantified for each sample. The median values are marked on the graph, and asterisks indicate statistical significance (Mann–Whitney, two-tailed).

Article Snippet: Antibodies used were: S9.6 (Kerafast ENH001), double-stranded DNA (dsDNA; Novus NBP3-07302), γH2AX (Abcam ab2893), EXO1 (Novus NBP2-16391), and CHAF1A (Cell Signaling Technology 5480s).

Techniques: Over Expression, Two Tailed Test, Immunofluorescence, Knock-Out, MANN-WHITNEY