FANCJ helicase promotes DNA end resection by facilitating CtIP recruitment to DNA double-strand breaks (original) (raw)
Fig 1
FANCJ facilitates DNA end resection.
(A) To measure end resection by BrdU staining, ER-_Asi_SI U2OS cells pre-labelled with BrdU for 24 h were depleted for the indicated proteins followed by treatment with 300 nM 4-OHT for 4 h or mock treated. The efficiency of knockdown of individual proteins was examined by western blotting (S1C Fig). Cells were fixed and stained with BrdU antibodies in native conditions to selectively detect ssDNA generated by end resection. CENP-F was used as an S/G2 phase marker to specifically examine DNA end resection during HR. Representative image for BrdU foci is shown. (B) Graph represents mean BrdU intensity from indicated samples in (A). N = 3; error bars indicate standard deviation (SD) and statistical significance was measured by two-tailed _Student_’s t-test of unequal variance. *p < 0.05; **p < 0.01; ***p < 0.001; N.S., non-significant. (C) ER-_Asi_SI U2OS cells depleted of the indicated proteins were treated with 300 nM 4-OHT for 4 h or mock treated. Cells were fixed and stained with γ-H2AX and pRPA2 (S4/S8) antibodies to detect ssDNA generated by end resection. Representative image for γ-H2AX and pRPA2 (S4/S8) foci are shown. (D) Graph represents the mean fluorescence intensity of γ-H2AX and pRPA2 (S4/S8) foci/nucleus from indicated cells in (C). N = 3; error bars indicate standard deviation (SD) and statistical significance was measured by two-tailed _Student_’s t-test of unequal variance. *p < 0.05; **p < 0.01; ***p < 0.001; N.S., non-significant. (E) ER-_Asi_SI U2OS cells treated with either control shRNA, shFANCJ #1 or shCtIP were treated with increasing dose of 4-OHT (0,100 and 400 nM) for 4 h. Whole cell lysates were resolved on 10% SDS-PAGE and probed for the indicated proteins to measure their damage induced enrichment in the cell. (F) ER-_Asi_SI U2OS cells depleted for the indicated proteins were treated with 300 nM 4-OHT for 4 h or mock treated. Cells were fixed and stained with γ-H2AX and RAD51 antibodies. Representative image for γ-H2AX and RAD51 foci are shown. (G) Graph represents the mean fluorescence intensity of γ-H2AX and RAD51 foci/nucleus from indicated cells in (F). N = 3; error bars indicate standard deviation (SD) and statistical significance was measured by two-tailed _Student_’s t-test of unequal variance. *p < 0.05; **p < 0.01; ***p < 0.001; N.S., non-significant. (H) ER-_Asi_SI U2OS cells depleted for the indicated proteins were treated with 300 nM 4-OHT for 4 h or mock treated. Cells were fixed and stained with γ-H2AX and RPA70 antibodies. Representative image for γ-H2AX and RPA70 foci are shown. (I) Graph represents the mean fluorescence intensity of γ-H2AX and RPA70 foci/nucleus from indicated cells in (H). N = 3; error bars indicate standard deviation (SD) and statistical significance was measured by two-tailed _Student_’s t-test of unequal variance. *p < 0.05; **p < 0.01; ***p < 0.001; N.S., non-significant. (J) Measurement of DSB end resection in ER-_Asi_SI U2OS cells transfected with control shRNA or shRNAs directed against BRCA1, FANCJ, CtIP, MRE11, DNA2 and 53BP1 as indicated using the assay established in S1A Fig. The efficiency of knockdown of individual proteins was examined by western blotting (S1C Fig). ER-_Asi_SI U2OS cells depleted of indicated proteins were synchronized in S/G2 phase as depicted in S1B Fig followed by treatment with 300 nM 4-OHT for 4 h or mock treated, genomic DNA (gDNA) was extracted and digested or mock digested with _Ava_I, _Nme_AIII, _Bsr_GI, _Bam_HI or _Hin_dIII overnight. DNA end resection adjacent to DSB1, DSB2 and No DSB site was measured by qPCR as described in ‘Materials and Methods’ section. N = 3; with error bars indicating SD and statistical significance was measured by two-tailed _Student_’s t-test of unequal variance. Summary of the % DSBs at the two selected _Asi_SI sites are shown in S2 Table. *p < 0.05; **p < 0.01; ***p < 0.001; N.S., non-significant.