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. 2022 May 16;11:e74700. doi: 10.7554/eLife.74700

Figure 1. RPA is loaded onto ssDNA after DSBs in G0 mammalian cells.

(A) Flow cytometric analysis of chromatin-bound RPA in G0-arrested Lig4-/- and Lig4-/-:Trp53bp1-/- abl pre-B cells before and 3 hr after 20 Gray IR. Representative of three independent experiments. (B) Flow cytometric analysis of chromatin-bound RPA before and 2 hr after 15 Gy IR in G0-arrested Lig4-/- abl pre-B cells (left), Lig4-/- cells depleted of MRE11 (middle), and Lig4-/- cells depleted of CtIP (right). Representative of three independent experiments. (C) Representative images and quantification of IR-induced RPA foci from three independent experiments in G0-arrested MCF10A cells before and 3 hr after 10 Gray IR. n=365 cells in -IR and n=433 cells in +IR. Red bars indicate average number of RPA foci in - IR = 0.96 and average number of RPA foci in +IR = 9.4 (****p<0.0001, unpaired t test). (D) RPA ChIP-seq tracks at AsiSI DSBs on chromosome 2, 5, and 4 at 4 hr (top) and 8 hr (bottom) after AsiSI endonuclease induction in G0-arrested Lig4-/- abl pre-B cells. (E) Representative END-Seq tracks showing resection at AsiSI DSBs at chromosome 2, 5, and 4 at 4 hr (top) and 8 hr (bottom) after AsiSI induction in G0-arrested Lig4-/- abl pre-B cells. END-seq data is representative from two independent experiments.

Figure 1.

Figure 1—figure supplement 1. RPA is loaded onto ssDNA after DSBs in G0 mammalian cells.

Figure 1—figure supplement 1.

(A) Flow cytometric analysis of cycling and STI treated Lig4-/- abl pre-B cells for BrdU content (y-axis) and DNA content (7-AAD, x-axis). (B) Flow cytometric analysis of chromatin-bound RPA in wild-type G0-arrested abl pre-B cells before and 3 hr after 20 Gray IR. (C) Western blot of bulk CtIP and MRE11 knockout in Lig4-/- abl pre-B cells. (D) Flow cytometric analysis of chromatin-bound RPA in wild-type G0-arrested abl pre-B cells before and 3 hr after 20 Gray IR in cells depleted of Rosa26 (control) and Artemis, which is encoded by the Dclrelc gene. (E) Flow cytometric analysis of chromatin-bound RPA loading in wild-type G0-arrested MCF10A cells before and 3 hr after 20 Gray IR. (F) Heat maps of RPA ChIP-seq results at top 200 AsiSI sites in G0-arrested Lig4-/- abl pre-B cells 4 hr (left) and 8 hr (right) after AsiSI-endonuclease induction.
Figure 1—figure supplement 1—source data 1. Original western blots for Figure 1.
Figure 1—figure supplement 2. The vast majority of AsiSI sites that are cleaved in G0 cells are in close proximity to the transcription start site of actively transcribed genes.

Figure 1—figure supplement 2.

(A) Frequency of broken AsiSI sites versus distance from transcription start sites (TSS). (B) Percentage of broken AsiSI sites within 2000bp of transcription start sites (TSS) and within transcriptionally active genes. (C) Proximity to TSS and level of gene expression for each of the AsiSI sites efficiently cleaved in G0 cells, from which the pie charts in (B) were generated.