Histone supply regulates S phase timing and cell cycle progression.
Bottom Line: During DNA replication, nucleosomes are disrupted and re-assembled with newly synthesized histones and DNA.We used a histone mutation of Drosophila melanogaster to show that histone supply levels, provided by a defined number of transgenic histone genes, regulate the length of S phase during the cell cycle.Lack of de novo histone supply not only extends S phase, but also causes a cell cycle arrest during G2 phase, and thus prevents cells from entering mitosis.
Affiliation: Abteilung Molekulare Entwicklungsbiologie, Max-Planck-Institut für biophysikalische Chemie, Göttingen, Germany Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, United Kingdom.Show MeSH
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Mentions: Mutations in the Drosophila ortholog of Chk1 (GRP) show developmental defects prior to cell cycle 15, excluding genetic experiments as we performed for lok (Fogarty et al., 1994; Su et al., 1999). Thus, we tested ATR/Chk1 checkpoint activation by using a phosphospecific antibody that recognizes the ATR-dependent phosphorylation of S345 in human Chk1 in response to replicative stress, for example, UV irradiation or HU treatment (Zhao and Piwnica-Worms, 2001). This antibody is expected to cross-react with Drosophila GRP due to sequence similarity and detected a single band in Western blots (Figure 5—figure supplement 1) as well as a clear signal in immunofluorescence (Figure 5—figure supplement 2) upon HU treatment of Drosophila S2R+ tissue culture cells. To test whether the ATR/Chk1 checkpoint is functional in HisC mutant embryos, we irradiated embryos with UV light (254 nm, UVC), which induces replication stress and replication fork uncoupling (Byun et al., 2005; Cimprich and Cortez, 2008). Wild type embryos and HisC mutant embryos accumulated phosphorylated GRP protein (pGRP) in response to UVC, showing that the checkpoint response is functional in the mutant embryos (Figure 5A–D). Without UVC treatment HisC mutant embryos did not display elevated pGRP levels as compared to wild type (Figure 5E–H), which was also verified by Western blotting of extracts from sorted HisC mutant and wild type sibling embryos (Figure 5I). In addition, treatment of HisC mutant embryos with the ATR inhibitor VE-821 (Prevo et al., 2012) or the Chk1 inhibitor CHIR-124 (Tse et al., 2007) did not result in a release of the cell cycle arrest (Figure 5—figure supplements 3 and 4). In addition to DSBs, replicative stress can induce phosphorylation of H2Av (Figure 4—figure supplement 1K–P), either directly by ATR dependent phosphorylation (Ward and Chen, 2001; Joyce et al., 2011) or through interconversion of single-stranded DNA generated at stalled replication forks into DSBs (Cimprich and Cortez, 2008). Consistent with the notion that the DNA damage checkpoints are functional in HisC mutant embryos we found accumulation of γH2Av in UVC-treated embryos to levels well above the background levels detected in untreated HisC mutant embryos in late S15 (Figure 5—figure supplement 5). Interestingly, UVC-treated embryos were able to enter M15 while displaying levels of γH2Av comparable or above to what we observed in untreated HisC mutant embryos (Figure 5—figure supplement 6). Taken together, these results strongly suggest that HisC mutant cells complete DNA replication in S phase without inducing significant DNA damage or replication stress and that the cell cycle arrest at the G2/M transition in HisC mutant cells is not mediated by the conventional S phase checkpoints.10.7554/eLife.02443.013Figure 5.HisC mutant cells do not activate the ATR/Chk1 DNA damage checkpoint.
Affiliation: Abteilung Molekulare Entwicklungsbiologie, Max-Planck-Institut für biophysikalische Chemie, Göttingen, Germany Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, United Kingdom.