Supplementary MaterialsFlow Cytometry Reporting Overview. the destiny of unresolved recombination intermediates that occur in k/o cell range produced from 293 cells using CRISPR/Cas934 (Fig. 1a). These resolvase-deficient cells exhibited a lower life expectancy regularity of sister chromatid exchanges (SCEs) weighed against cells, or MUS81-depleted regular cells (Supplementary Fig. 1a). These data concur that resolvases are in charge of producing buy THZ1 crossovers17, 28C31. Open up in another window Body 1 Phenotypic analysis of resolvase-deficient cells.(a) Schematic diagram depicting the experimental system. (b) 293 cells and cells were treated with control siRNA or siRNA against MUS81 for 96 h. FACS analyses show their DNA content distributions. (c) Quantification of G1, S and G2 populations of cells treated as in (b). (d) Cells were treated as in (b) and stained with buy THZ1 cyclin B antibody (upper panel) or histone H3 pSer10 antibody (lower panel). Percentages of cyclin B-positive and histone H3 pSer-positive cells were quantified. (e) Clonogenic cell survival assays were carried out on 293 cells and cells treated with control siRNA or siRNA against MUS81. Complementation by stable expression of GEN1-3xFLAG is usually indicated. The survival of control siRNA-treated 293 cells is usually defined as 100%. (f) Clonogenic cell survival assays were carried out on 293 and cells treated with control siRNA or siRNA against MUS81, and the indicated concentrations of cisplatin (Cis-Pt). (g) Chromosome segmentation in a metaphase spread from cells treated with siRNA against MUS81 and a brief cisplatin treatment, and released into fresh media for 24 h. (h) 293 cells and cells were treated as in (g). 75 metaphase spreads per condition were analysed for chromosome segmentation. (i) and cells expressing GEN1, RusAWT-GEN1 or RusAD70N-GEN1 were treated as in (g). 60 metaphase spreads per condition were analysed for chromosome segmentation. In b and g, representative data from three impartial experiments are shown. Quantified data in c-f, we and h represent the mean s.d. of n = 3 indie experiments. Supply data can be purchased in Supplementary Desk 1. P beliefs had been determined utilizing a two-tailed t-test. The resolvase-deficient cells uncovered some stunning phenotypic properties. First of all, we noticed a build up of cells with 4N DNA articles (Fig. 1b,c). To verify G2 arrest, cells had been treated with antibodies against cyclin B (a G2 marker) and histone H3 pSer10 (a mitotic marker), and analysed by FACS (Fig. 1d). A substantial upsurge in cyclin B-positive cells, however, not histone H3 pSer10-positive cells was noticed. G2 arrest happened 96 hours after MUS81 siRNA treatment of the cells (Supplementary Fig. 1b), indicating the deposition of endogenous DNA harm. Furthermore, clonogenic assays demonstrated massive artificial lethality ( 10% cell survival) (Fig. 1e). Loss of viability and G2 arrest were rescued by exogenous expression of FLAG-tagged GEN1 (Fig. 1e and Supplementary Fig. 1c,d). The resolvase-deficient cells were highly sensitive to the DNA damaging brokers cisplatin and camptothecin (CPT) (Fig. 1f and Supplementary Fig. 1e), but only mildly sensitive to replication stress induced by aphidicolin (APH) (Supplementary Fig. 1f). These results are consistent with the involvement of MUS81-EME1 and GEN1 in the resolution of DNA repair intermediates. To gain further insights into the interplay between GEN1 and components of the SMX complex (in particular MUS81-EME1 and SLX1-SLX4), cells co-depleted for MUS81 (Supplementary Fig. buy THZ1 2c,d). The conversation of MUS81 with the SLX4 scaffold buy THZ1 protein is known to be critical for its resolution functions27, 30, 31, 35. We therefore mutated the key conserved residues in SLX4 (E1577A, L1578A) equivalent to those previously recognized in mouse SLX4 that abolish MUS81-SLX4 interactions30 (Supplementary Fig. 2e), and observed that depletion of GEN1 from SLX4E1577A L1578A (SLX4ELAA) cells induced cell death and cell cycle arrest Cdh13 (Supplementary Fig. 2f-h). These results confirm the synthetic relationship between GEN1 and MUS81/SLX4. Unresolved recombination intermediates form ultra-fine bridges To investigate the consequences of mitosis with unresolved recombination intermediates, we briefly treated resolvase-deficient cells with cisplatin and prepared metaphase spreads 24h later. We observed tightly-associated sister chromatids that exhibited a segmented appearance (Fig. 1g,h). This unusual morphology was previously attributed to defects in chromosome condensation at sites of sister chromatid entanglements17, 29, 31. Elevated levels of chromosome segmentation were observed even in the absence of exogenous damage (Supplementary.