Fig. 2 | Nature Communications

Fig. 2

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

Fig. 2

FAAP24 and MHF are important for recruiting FANCM to Flex1 to prevent DSB formation and mitotic recombination. a DSB formation at Flex1 or Luc surrounding regions was analyzed by ChIP analysis of pCEP4-Flex1 or pCEP4-Luc10 using H2AX-S139p (γH2AX) antibody. Anti-γH2AX ChIP was performed in U2OS cells expressing FANCM shRNA (left), expressing Flag-FANCM-WT or K117R with endogenous FANCM silenced by shRNA (middle), or expressing FAAP24 shRNA with or without HU treatment (2 mM, 6 h) (right). Western blots for the expression of FANCM, Flag-FANCM WT and K117R, and FAAP24 are shown in Supplementary Fig. 5b. b Flag-FANCM or empty vector (Ctrl) was expressed in U2OS (HR-Flex) reporter cell line with Flex1 stably integrated into the genome. Anti-Flag ChIP was performed using indicated primer sets (P1 or P2), or primers corresponding to GAPDH (left); or using P1 before or after APH treatment (0.4 μM, 24 h) (right). ChIP value in Ctrl is set up as 1 for normalization. c Anti-Flag ChIP at Flex1 was performed in U2OS (HR-Flex) cells expressing Flag-FANCM and shRNAs for FAAP24 (left) or MHF1 (right) with vector as a control. Western blots for the expression of FAAP24 and MHF1 are shown in Supplementary Fig. 5d. d Spontaneous recombination was assayed in U2OS (HR-Flex) cells expressing shRNA for FAAP24 (left) or MHF1 (right) with a vector control. e Anti-Flag ChIP at Flex1 was performed in U2OS (HR-Flex) cells expressing Flag-FANCM-WT or indicated mutants with the vector as a control. Western blots showing FANCM expression are in Supplementary Fig. 5e. f Anti-Flag ChIP at Flex1 was performed in U2OS (HR-Flex) cells expressing Flag-FAAP24 WT or mutants (left), or Flag-FANCM and HA-FAAP24 WT or indicated FAAP24 mutants with endogenous FAAP24 silenced by shRNA (right). ChIP in the vector expressing cells serves as the control. DB DNA binding mutant: K171A/K173A; MB FANCM binding mutant: V198A/V199A/G200A. Western blots showing relevant protein expression are in Supplementary Fig. 5f, g. g Spontaneous recombination was assayed in U2OS (HR-Flex) cells expressing Flag-FAAP24 WT and mutant alleles with endogenous FAAP24 silenced by shRNA