Requirement for Heterochromatin Stability Figure 6. DNA repair checkpoint and mitotic checkpoint proteins are essential for the viability of Su(var)3-9 mutants. A) The chart lists the viability of the double mutants of Su(var)3-9 with mutations in the DNA damage checkpoint and mitotic checkpoint pathway. Viability was calculated relative to single homozygous checkpoint mutants, which are less viable than Su(var)3-9 single mutants. Progeny counts are in Table S1. P values were calculated by the Chi-square test. B) Cell cycle analysis of wild type, Su(var)3-9, grp; Su(var)3-9 and lok; Su(var)3-9 mutant imaginal discs and brains. The percentages of G1 cells in the two double mutants were higher than wild type and single Su(var)3-9 mutants. The percentages of S phase cells in the two double mutants are lower than wild type, but do not differ from Su(var)3-9. The percentages of G2 cells in the double mutants are lower than wild type and Su(var)3-9. The mitotic indices in the double mutants were lower than Su(var)3-9 and do not significantly differ from the wild type. P,0.05 for all tests that show significant differences between wild type and Su(var)3-9; p values were calculated by Student’s t test, and n.1000 cells for each genotype. doi:10.1371/journal.pgen.1000435.g006 Figures S2A and B). Foci localized to DAPI-bright regions in dcr-2 increased 24-fold over wild type for cH2Av and 33-fold for TUNEL (Figure S2C and D). Foci in DAPI-weak regions do not differ significantly between wild type and dcr-2. Therefore, increased DNA damage in dcr-2 occurs in heterochromatic DNAs of dcr-2 mutant cells. IF analyses showed that cH2Av foci in dcr-2 mutant oocytes increased over wild type by 1.5- (foci number) to 3- fold (volumetric analysis). In both meiotic and mitotic cells, the increases in spontaneous DNA damage were significant, but less severe than in Su(var)3-9 mutants (Figure S3). Su(var)3-9 mutant cells contained elevated frequencies of damage in all interphase cell cycle stages (Figure 5B). However, gH2Av foci enrichment in dcr-2 mutant cells only occurred during S phase (Figure 7A), suggesting that increased DNA breaks are repaired in S phase and do not persist into G2. As observed for Su(var)3-9, dcr-2 mutants displayed reduced embryonic viability, but developed normally in larval and pupal stages. The percentages of dcr-2 cells in G2, mitosis, and apoptosis also increased relative to wild type (Figure 7B). However, viability analysis of double mutant flies showed that mutations in DNA PLoS Genetics | www.plosgenetics.org 8 March 2009 | Volume 5 | Issue 3 | e1000435