Fig. 5 | Nature Communications

Fig. 5

From: The concerted roles of FANCM and Rad52 in the protection of common fragile sites

Fig. 5

Rad52 plays a critical role in repairing DSBs at Flex1. a, b Spontaneous recombination was determined in U2OS (HR-Flex) cells expressing shRNAs for Rad51 or Rad52, or control vector, 6 or 12 days after expression of FANCM shRNA or control vector (a), or 4 days after HU treatment (2 mM, 24 h) or without (b). c Anti-Flag ChIP at Flex1 was performed in U2OS (HR-Flex) cells expressing Flag-Rad51 (left) and Flag-Rad52 (right). The expression of indicated protein is shown by western blots. d ChIP analysis of γH2AX at Flex1 or Luc was performed in U2OS cells containing pCEP4-Flex1 or pCEP4-Luc plasmids and expressing shRNAs for FANCM, Rad52 or both after treatment of HU (2 mM, 6 h). The ChIP signal of γH2AX is quantified by relative fold to the Ctrl. e Schematic drawing of HR reporters containing the I-SceI site (HR), Flex1 with the I-SceI site (HR-Flex), and Luc with the I-SceI site (HR-Luc) is shown (top). The homology of the I-SceI cleaved EGFP cassettes to the donor template (D-EGFP) is indicated as white boxes. I-SceI-induced HR was assayed in U2OS HR, HR-Flex, and HR-Luc reporter cell lines with expression of Rad52 shRNA or control vector (bottom). Rad52 expression is shown by western blot analysis. f Schematic drawing of HR reporters HR-Flex/D-Flex and HR-Luc/D-Luc, which contain identical Flex1 and Luc sequences in both EGFP recipient cassettes (EGFP::Flex1/I-SceI and EGFP::Luc/I-SceI) and the donor templates (D-Flex or D-Luc) is shown on top. The BamHI and EcoRI sites in the donor templates are at the corresponding position of the I-SceI site in the recipient EGFP cassettes. U2OS (HR-Flex/D-Flex) or U2OS (HR-Luc/D-Luc) cells with or without expressing Rad52 shRNAs were infected with I-SceI lentiviruses and 3 days after, genomic DNA was extracted and digested with I-SceI, followed by PCR using indicated primers (shown as arrows). The percentage of BamHI and EcoRI digestible PCR products among total DNA was calculated. Predicted enzyme digestion patterns of parental EGFP recipient cassettes (EGFP::Flex1/I-SceI and EGFP::Luc/I-SceI) and the repair products generated by HR or imperfect end joining are shown in the box at right