Fig 2: Ectopic expression of miR-15a, miR-16 sensitizes breast cancer cells to DNA damage through BMI1 dependent ubiquitination pathway. Western blot analysis showing the expression of BMI1, RING1A, RING1B, EZH2, γ-H2AX, Ub-H2A, p-CHK2, p-ATM, RNF8, RNF168, MEL18, p53BP, BRCA1 proteins in MDAMB-231 cells transfected with miR-15a, miR-16 or both miR-15a/16 under etoposide-induced DNA damage conditions.. Actin served as a gel loading control. Blots were cropped to enhance the representation (A,B). Immunofluorescence data showing accumulation of BMI1, ϒ-H2AX (C) RING1B (D) and Ub-H2A (E) p53BP (F) in MDAMB-231 cells transfected with miR-15a miR-16 or both miR-15a/16 under etoposide- treated conditions. Bar indicates 200 μm.

Fig 3: Time-dependent expression of proteins after ectopic expression of miR-15a/16 and overexpression of miR-15a/16 impedes homologous recombination-mediated repair. Western blot analysis showing the expression of BMI1, γ-H2AX, Ub-H2A in MDAMB-231 cells transfected with both miR-15a/16 followed by treatment with etoposide for 2 to 72 hrs. Actin was used as a gel loading control. During the course of time periods (0 hrs to 72 hrs). Blots were cropped to enhance the representation (A). Heat map showing the endogenous expression of quantitative RT-PCR analysis. The Green colour in the heat map indicates decreased and red colour indicates increased mRNA expression level. Gene specific primers were used for the amplification. GAPDH was used as endogenous control (B,C) Metaphase chromosomes of cells overexpressing Mir-15a/16. (i) Metaphase fromcontrol, (ii) Metaphase from irradiated cell showing gaps (small arrow) and radials (large arrows)in human H1299 cells (D). In human H1299 cells S-phase specific chromosome aberrations after 2 Gy irradiations. *p < 0.05, **p < 0.01, ***p < 0.001 Student t-test (E) Homologous recombination (HR) repair of DNA DSB repair in human H1299 cells are shown with and without I-SceI induction.*p < 0.05, **p < 0.01, ***p < 0.001Student t-test (F).

Fig 4: L22P mice accumulate base excision repair (BER) intermediates and exhibit double strand breaks (DSBs). (A) Number of apurinic/apyrimidinic (AP) sites/105 nucleotides from DNA extracted from wild type (WT) and L22P mice stomach tissues; (B) Representative image of spontaneous single strand breaks (SSBs) from Alkali comet assay on WT and L22P mouse embryonic fibroblasts (MEFs) cells, All image captured with 63× objective of Zeiss fluorescence microscope (Zeiss, San Diego, CA, USA); (C) Measured tail moment for measuring spontaneous SSBs in WT and L22P MEFs cells and data analysis carried out using Student’s t test; (D) Immunohistochemical staining with phosphorylated H2AX (γH2AX) antibody of stomach tissue sectioned from WT and L22P mice; Image shows 5× magnification and were captured using Scanscope (Leica Biosystem, Vista, CA, USA); (E) Percent of γH2AX-positive cells in the WT (n = 2000) and L22P (n = 2078) mouse stomachs. Data were analyzed using Mann–Whitney test; (F) Western blot analysis of γH2AX in WT and L22P cells; Fold change calculated using the ratio of γH2AX band intensity to loading control of alpha-tubulin and normalized to wild type; (G) Western blot analysis for Chk1 and Chk1 phosphorylation at Ser 317; Fold change calculated using the ratio of band intensity of Chk1p to chk1 and normalized to wild type. (H) Representative image of the γH2AX and 53BP1 colocalization in WT and L22P MEFs; (I) Percent of cells showing colocalization of γH2AX and 53BP1 with Methyl methanesulfonate (MMS) DNA damage and recovery after treatment. L22P and WT MEFs were treated with 1.5 mM MMS and examined for the recovery of DSBs after removing MMS at the following times: 0 hours (WT n = 85; L22P n = 147); 6 hours (WT n = 106 versus L22P n = 108); 12 hours (WT n = 89 versus L22P n = 168); and 24 hours (WT n = 95 versus L22P n = 101). Note that all images were taken at 63× with a Zeiss fluorescence microscope (Zeiss, San Diego, CA, USA) with a constant exposure time, and any cell with >5 foci of γH2AXcolocalize with 53BP1/cell was categorized as a positive. Data were analyzed using two-way ANOVA test. Significant differences are designated by *** p < 0.001; **** p < 0.0001 using GraphPad Prism (Prism 7.00 for Mac, GraphPad Software, San Diego, CA, USA).

Fig 5: 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