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• W1569124576 abstract "•Replication initiation triggers the rapid decondensation of chromatids•Replication initiation counteracts condensins, inactive MCM-2–7, and MEL-28•DNA replication promotes condensin II accumulation and chromosome condensation During cell division, chromatin alternates between a condensed state to facilitate chromosome segregation and a decondensed form when DNA replicates. In most tissues, S phase and mitosis are separated by defined G1 and G2 gap phases, but early embryogenesis involves rapid oscillations between replication and mitosis. Using Caenorhabditis elegans embryos as a model system, we show that chromosome condensation and condensin II concentration on chromosomal axes require replicated DNA. In addition, we found that, during late telophase, replication initiates on condensed chromosomes and promotes the rapid decondensation of the chromatin. Upon replication initiation, the CDC-45-MCM-GINS (CMG) DNA helicase drives the release of condensin I complexes from chromatin and the activation or displacement of inactive MCM-2–7 complexes, which together with the nucleoporin MEL-28/ELYS tethers condensed chromatin to the nuclear envelope, thereby promoting chromatin decondensation. Our results show how, in an early embryo, the chromosome-condensation cycle is functionally linked with DNA replication. During cell division, chromatin alternates between a condensed state to facilitate chromosome segregation and a decondensed form when DNA replicates. In most tissues, S phase and mitosis are separated by defined G1 and G2 gap phases, but early embryogenesis involves rapid oscillations between replication and mitosis. Using Caenorhabditis elegans embryos as a model system, we show that chromosome condensation and condensin II concentration on chromosomal axes require replicated DNA. In addition, we found that, during late telophase, replication initiates on condensed chromosomes and promotes the rapid decondensation of the chromatin. Upon replication initiation, the CDC-45-MCM-GINS (CMG) DNA helicase drives the release of condensin I complexes from chromatin and the activation or displacement of inactive MCM-2–7 complexes, which together with the nucleoporin MEL-28/ELYS tethers condensed chromatin to the nuclear envelope, thereby promoting chromatin decondensation. Our results show how, in an early embryo, the chromosome-condensation cycle is functionally linked with DNA replication. Cell-cycle progression requires the ordered succession of cell-cycle stages, and checkpoints ensure that critical cell-cycle events such as DNA replication or chromosome alignment are completed before subsequent stages can occur. Changes in cyclin-dependent kinase (CDK) kinase activity and differential cyclin association drive major transitions such as the initiation of S phase, mitosis, and the subsequent segregation of chromatids. Faithful chromosome segregation requires the structural reorganization of chromosomes into condensed metaphase chromosomes, which is needed for the segregation of chromatids during anaphase. Conversely, chromosome decondensation facilitates transcription and DNA replication. In rapidly dividing embryos, S phase and mitosis alternate without apparent G1 or G2 phases. Thus, decondensation, DNA replication, and re-condensation occur in a short period and could potentially overlap. Indeed, we know little about how DNA replication and chromatin condensation and decondensation are coordinated. Condensation is mediated by condensin complexes, pentameric ring-shaped structures composed of two structural maintenance of chromosomes (SMC) subunits that exhibit ATPase activity and that are related to cohesin subunits, plus three regulatory units known as chromosome associated proteins (CAPs). Most organisms contain two condensin complexes, condensin I and II, which share the same SMC units (MIX-1/SMC2 and SMC-4 in the worm) but differ in their regulatory subunits, termed CAPG-1, DPY-26, and DPY-28 for C. elegans condensin I and CAPG-2, KLE-2, and HCP-6 for C. elegans condensin II (Csankovszki et al., 2009Csankovszki G. Collette K. Spahl K. Carey J. Snyder M. Petty E. Patel U. Tabuchi T. Liu H. McLeod I. et al.Three distinct condensin complexes control C. elegans chromosome dynamics.Curr. Biol. 2009; 19: 9-19Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, Hirano, 2012Hirano T. Condensins: universal organizers of chromosomes with diverse functions.Genes Dev. 2012; 26: 1659-1678Crossref PubMed Scopus (245) Google Scholar, Piazza et al., 2013Piazza I. Haering C.H. Rutkowska A. Condensin: crafting the chromosome landscape.Chromosoma. 2013; 122: 175-190Crossref PubMed Scopus (29) Google Scholar, Thadani et al., 2012Thadani R. Uhlmann F. Heeger S. Condensin, chromatin crossbarring and chromosome condensation.Curr. Biol. 2012; 22: R1012-R1021Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). In vertebrates, condensin I is cytoplasmic during interphase and appears to stabilize chromosome rigidity after nuclear envelope breakdown (Hirota et al., 2004Hirota T. Gerlich D. Koch B. Ellenberg J. Peters J.M. Distinct functions of condensin I and II in mitotic chromosome assembly.J. Cell Sci. 2004; 117: 6435-6445Crossref PubMed Scopus (270) Google Scholar, Ono et al., 2004Ono T. Fang Y. Spector D.L. Hirano T. Spatial and temporal regulation of Condensins I and II in mitotic chromosome assembly in human cells.Mol. Biol. Cell. 2004; 15: 3296-3308Crossref PubMed Scopus (274) Google Scholar). Condensin II is nuclear, is required for sister chromatid resolution during S phase, and promotes chromosomal axis formation during prophase (Cuvier and Hirano, 2003Cuvier O. Hirano T. A role of topoisomerase II in linking DNA replication to chromosome condensation.J. Cell Biol. 2003; 160: 645-655Crossref PubMed Scopus (90) Google Scholar, Ono et al., 2013Ono T. Yamashita D. Hirano T. Condensin II initiates sister chromatid resolution during S phase.J. Cell Biol. 2013; 200: 429-441Crossref PubMed Scopus (66) Google Scholar). In C. elegans embryos, condensin II is required for condensation during prophase and is concentrated on chromosomal axes (Csankovszki et al., 2009Csankovszki G. Collette K. Spahl K. Carey J. Snyder M. Petty E. Patel U. Tabuchi T. Liu H. McLeod I. et al.Three distinct condensin complexes control C. elegans chromosome dynamics.Curr. Biol. 2009; 19: 9-19Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, Hagstrom et al., 2002Hagstrom K.A. Holmes V.F. Cozzarelli N.R. Meyer B.J. C. elegans condensin promotes mitotic chromosome architecture, centromere organization, and sister chromatid segregation during mitosis and meiosis.Genes Dev. 2002; 16: 729-742Crossref PubMed Scopus (262) Google Scholar, Kaitna et al., 2002Kaitna S. Pasierbek P. Jantsch M. Loidl J. Glotzer M. The aurora B kinase AIR-2 regulates kinetochores during mitosis and is required for separation of homologous Chromosomes during meiosis.Curr. Biol. 2002; 12: 798-812Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Worm condensin I is cytoplasmic, localizes to chromosomes after nuclear envelope breakdown, and appears to be required for chromosome segregation (Csankovszki et al., 2009Csankovszki G. Collette K. Spahl K. Carey J. Snyder M. Petty E. Patel U. Tabuchi T. Liu H. McLeod I. et al.Three distinct condensin complexes control C. elegans chromosome dynamics.Curr. Biol. 2009; 19: 9-19Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). In C. elegans, a third condensin complex functions in dosage compensation for sex chromosomes (Meyer, 2010Meyer B.J. Targeting X chromosomes for repression.Curr. Opin. Genet. Dev. 2010; 20: 179-189Crossref PubMed Scopus (90) Google Scholar). Recent evidence suggests that condensin rings encircle DNA (Cuylen et al., 2011Cuylen S. Metz J. Haering C.H. Condensin structures chromosomal DNA through topological links.Nat. Struct. Mol. Biol. 2011; 18: 894-901Crossref PubMed Scopus (152) Google Scholar, Cuylen et al., 2013Cuylen S. Metz J. Hruby A. Haering C.H. Entrapment of chromosomes by condensin rings prevents their breakage during cytokinesis.Dev. Cell. 2013; 27: 469-478Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), and chromosome compaction might involve the entrapment of more than one DNA molecule or the interaction of condensin rings (for review, see Thadani et al., 2012Thadani R. Uhlmann F. Heeger S. Condensin, chromatin crossbarring and chromosome condensation.Curr. Biol. 2012; 22: R1012-R1021Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Eukaryotic DNA replication is divided into two non-overlapping phases (Blow and Dutta, 2005Blow J.J. Dutta A. Preventing re-replication of chromosomal DNA.Nat. Rev. Mol. Cell Biol. 2005; 6: 476-486Crossref PubMed Scopus (531) Google Scholar, DePamphilis et al., 2006DePamphilis M.L. Blow J.J. Ghosh S. Saha T. Noguchi K. Vassilev A. Regulating the licensing of DNA replication origins in metazoa.Curr. Opin. Cell Biol. 2006; 18: 231-239Crossref PubMed Scopus (140) Google Scholar). In late mitosis and early G1, replication origins are licensed for replication by loading Mcm2–7 double hexamers, which requires the loading factors ORC, Cdc6, and Cdt1. During S phase, CDKs and Dbf4-dependent kinases activate the Mcm2‐7 helicase by promoting its interaction with Cdc45 and the GINS complex (Gambus et al., 2006Gambus A. Jones R.C. Sanchez-Diaz A. Kanemaki M. van Deursen F. Edmondson R.D. Labib K. GINS maintains association of Cdc45 with MCM in replisome progression complexes at eukaryotic DNA replication forks.Nat. Cell Biol. 2006; 8: 358-366Crossref PubMed Scopus (593) Google Scholar, Ilves et al., 2010Ilves I. Petojevic T. Pesavento J.J. Botchan M.R. Activation of the MCM2-7 helicase by association with Cdc45 and GINS proteins.Mol. Cell. 2010; 37: 247-258Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar, Moyer et al., 2006Moyer S.E. Lewis P.W. Botchan M.R. Isolation of the Cdc45/Mcm2-7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase.Proc. Natl. Acad. Sci. USA. 2006; 103: 10236-10241Crossref PubMed Scopus (544) Google Scholar). This CMG (Cdc45-MCM-GINS) helicase unwinds the template DNA, allowing for RPA (single-strand binding protein) binding and DNA synthesis by DNA polymerases. In C. elegans embryos, chromatin is licensed at the end of M phase; when nuclei form and chromatin decondenses, licensing factors are then exported from nuclei, thereby ending the licensing phase and preventing re-replication (Sonneville et al., 2012Sonneville R. Querenet M. Craig A. Gartner A. Blow J.J. The dynamics of replication licensing in live Caenorhabditis elegans embryos.J. Cell Biol. 2012; 196: 233-246Crossref PubMed Scopus (56) Google Scholar). The rapidly dividing early embryo has relatively weak cell-cycle checkpoints, which allow continued cycling even when essential processes, such as DNA replication, are defective (Brauchle et al., 2003Brauchle M. Baumer K. Gönczy P. Differential activation of the DNA replication checkpoint contributes to asynchrony of cell division in C. elegans embryos.Curr. Biol. 2003; 13: 819-827Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, Budirahardja and Gönczy, 2009Budirahardja Y. Gönczy P. Coupling the cell cycle to development.Development. 2009; 136: 2861-2872Crossref PubMed Scopus (78) Google Scholar, Encalada et al., 2000Encalada S.E. Martin P.R. Phillips J.B. Lyczak R. Hamill D.R. Swan K.A. Bowerman B. DNA replication defects delay cell division and disrupt cell polarity in early Caenorhabditis elegans embryos.Dev. Biol. 2000; 228: 225-238Crossref PubMed Scopus (104) Google Scholar). Cell-cycle analysis is further facilitated by the rapid turnover of cytosolic proteins in the gonad (Oegema and Hyman, 2006Oegema K. Hyman A.A. Cell division.WormBook. 2006; 2006: 1-40Google Scholar) and the extended period of time needed for progressing from the premeiotic S phase through the extended meiotic prophase (Jaramillo-Lambert et al., 2007Jaramillo-Lambert A. Ellefson M. Villeneuve A.M. Engebrecht J. Differential timing of S phases, X chromosome replication, and meiotic prophase in the C. elegans germ line.Dev. Biol. 2007; 308: 206-221Crossref PubMed Scopus (150) Google Scholar), which allows depletion of replication genes before the first embryonic S phase without affecting the previous, pre-meiotic S phase (see below). Here, we investigate the functional relationship between DNA replication and the chromosome-condensation cycle by combining RNAi and in vivo imaging in C. elegans embryos. We show that replication commences concomitant with decondensation and that replication initiation, but not elongation, promotes decondensation. We provide evidence that replication initiation is needed for the dissociation of the MEL-28 (ELYS) nucleoporin from chromatin during chromosome decondensation and that MEL-28 depletion rescues the decondensation defect associated with blocking replication initiation. Finally, we show that genome duplication is required for condensin II concentration on chromosomal axes and for proper chromatin condensation in prophase. Our results reveal that DNA replication and the chromosome-condensation cycles are tightly coupled. We used RNAi and in vivo imaging of C. elegans embryos to uncover the links between DNA replication and the chromosome-condensation cycle. We visualized chromosomal DNA by employing mCherry fused to histone 2B (H2B) (mCherry-H2B) and the nuclear envelope by using the nucleoporin NPP-9 fused to GFP. Figure 1A and Movie S1, where the entire embryo is displayed, show the sequence of events occurring from meiotic anaphase II to the end of the first embryonic cell cycle. Shortly after fertilization, oocyte-derived chromosomes, which we will refer to as female chromosomes, complete the two meiotic divisions (meiosis I and II) leading to the extrusion of the two polar bodies and the formation of a haploid female nucleus. At the end of anaphase II, a ring of GFP-NPP-9 forms around the female chromosomes, followed by rapid decondensation of the chromatin. The female and male haploid nuclei grow in size at opposite poles of the embryo concomitant with the bulk of DNA replication (Edgar and McGhee, 1988Edgar L.G. McGhee J.D. DNA synthesis and the control of embryonic gene expression in C. elegans.Cell. 1988; 53: 589-599Abstract Full Text PDF PubMed Scopus (141) Google Scholar). These nuclei then migrate toward each other and meet at the posterior half of the cell and then move to the center of the cell. Concomitant with nuclear migration, chromatin patches indicative of condensation form and distinct chromosomes become progressively apparent. Upon nuclear disassembly, indicated by the disappearance of nucleoporins, chromosomes congress on the metaphase plate and anaphase ensues (Figure 1A; Movie S1). Within the gonad of an adult worm, the differentiation of a mature oocyte from a mitotic germ cell takes >24 hr (Jaramillo-Lambert et al., 2007Jaramillo-Lambert A. Ellefson M. Villeneuve A.M. Engebrecht J. Differential timing of S phases, X chromosome replication, and meiotic prophase in the C. elegans germ line.Dev. Biol. 2007; 308: 206-221Crossref PubMed Scopus (150) Google Scholar), a period of time sufficient to inactivate genes by RNAi in the embryo without affecting the premeiotic S phase. As a first step to assess the relationship between DNA replication and chromosome condensation, we used RNAi to deplete the licensing factor MCM-7. The assembly of hexameric MCM-2–7 onto chromosomes during anaphase requires the presence of all six MCM-2–7 subunits. We therefore used GFP-MCM-3 as a marker for MCM-2–7 chromatin loading. When MCM-7 was depleted, GFP-MCM-3 failed to load onto anaphase chromosomes or accumulate in nuclei during S phase, consistent with the inactivation of the MCM-2–7 complex (Sonneville et al., 2012Sonneville R. Querenet M. Craig A. Gartner A. Blow J.J. The dynamics of replication licensing in live Caenorhabditis elegans embryos.J. Cell Biol. 2012; 196: 233-246Crossref PubMed Scopus (56) Google Scholar) (Figure 1B). When mcm-7 was knocked down by RNAi, chromosome condensation was compromised during prophase of the first embryonic cell cycle and massive chromatin bridges were observed during anaphase (“cut phenotype”) (Figure 1B; Movie S2). In addition, we observed a delay in chromosome decondensation at the end of meiotic anaphase II (Figure 1B, S phase; Movie S2), a phenotype we will examine in more detail below. Such a cut phenotype has been associated with replication defects in fission yeast and human cells (Hirano et al., 1986Hirano T. Funahashi S. Uemura T. Yanagida M. Isolation and characterization of Schizosaccharomyces pombe cutmutants that block nuclear division but not cytokinesis.EMBO J. 1986; 5: 2973-2979Crossref PubMed Google Scholar, Samejima et al., 1993Samejima I. Matsumoto T. Nakaseko Y. Beach D. Yanagida M. Identification of seven new cut genes involved in Schizosaccharomyces pombe mitosis.J. Cell Sci. 1993; 105: 135-143PubMed Google Scholar, Steigemann et al., 2009Steigemann P. Wurzenberger C. Schmitz M.H. Held M. Guizetti J. Maar S. Gerlich D.W. Aurora B-mediated abscission checkpoint protects against tetraploidization.Cell. 2009; 136: 473-484Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar) and is thought to be a consequence of cells passing through mitosis with unreplicated DNA. As an aside, in MCM-7-depleted embryos, no chromatin bridges were observed during the meiotic anaphases (Figure 1B, anaphase II), suggesting that the pre-meiotic S phase was not affected. In order to show directly that MCM-7 depletion inhibits replication, we adapted 5-ethynyl-2′-deoxyuridine (EdU) labeling procedures to measure replication in the entire first embryonic cell cycle (see Experimental Procedures). Permeabilized embryos incubated with EdU throughout the first embryonic cell cycle showed a ∼90% reduction of EdU incorporation after one cell cycle when MCM-7 was depleted (Figures 1C and 1D). In agreement with compromised EdU incorporation, GFP-histone H2B intensity during the first mitosis of mcm-7 RNAi embryos was approximately half of wild-type, consistent with an almost complete block in replication during the first embryonic S phase (Figures 2B and 2H ). Therefore, during the first embryonic cell cycle, MCM-2–7 are required for replication, condensation, and segregation of the chromatin. We next wished to test if other replication genes are required for chromatin condensation. The majority of C. elegans genes have been systematically depleted by RNAi, and DIC (differential interference contrast) recordings of the first embryonic cell cycles have been deposited into “phenobank” (Sönnichsen et al., 2005Sönnichsen B. Koski L.B. Walsh A. Marschall P. Neumann B. Brehm M. Alleaume A.M. Artelt J. Bettencourt P. Cassin E. et al.Full-genome RNAi profiling of early embryogenesis in Caenorhabditis elegans.Nature. 2005; 434: 462-469Crossref PubMed Scopus (708) Google Scholar). We screened phenobank to identify replication genes whose inactivation led to a cut phenotype akin to MCM depletion. Examining knockout phenotypes of 40 genes expected to be involved in DNA replication, we found that 14 were associated with a first-cycle cut phenotype (Table S1). The absence of a cut phenotype in other replication mutants is consistent with carryover of maternal protein, partial RNAi depletion, or genetic redundancy. To further analyze regulatory connections between DNA replication and the chromosome condensation cycle, we focused on replication factors involved in various stages of replication whose depletion gave a first-cycle cut phenotype. We thus depleted these replication factors: the CDT-1 licensing factor, MCM-7, the CDC-45 initiation factor, the RPA-1 single-strand binding protein, the proliferating cell nuclear antigen (PCNA) ortholog PCN-1, and the RNR-1 ribonucleotide reductase required for dNTP supply. With the exception of rnr-1, each of these RNAi treatments caused a large reduction in EdU incorporation (Figures 2A and 2G). The EdU incorporation in embryos treated with rnr-1 RNAi can be explained by the depletion of the cellular dNTP pools, which favors the incorporation of EdU (itself not requiring ribonucleotide reductase) during residual replication. In addition, with the exception of rpa-1 and pcn-1, each of these RNAi treatments halved the intensity of chromatin bound GFP-H2B at first mitosis, consistent with a replication block (Figures 2B and 2H). The high GFP-H2B intensity in cells treated with rpa-1 and pcn-1 RNAi is surprising, but it may reflect an abnormal chromatin structure in cells lacking these factors. Recordings of GFP-H2B show that all of these depletions led to defective chromosome condensation at first mitosis (Figure 2B; Movie S3). Analysis of Hoechst-stained chromosomes (Figure 2C, left panels) of late prophase embryos, as defined by the position of the nuclei and by staining for phosphorylated Ser10 of histone H3 (Figure 2C), indicated that condensation was not completely abolished; chromatin patches formed during prophase but did not congress into single chromosomes. Such patches were much less discernible when GFP-H2B was imaged, likely due to the background of nucleoplasmic GFP-H2B. Defective condensation was followed by massive anaphase bridging (Figure 2B; Movie S3). Such condensation and segregation defects resemble the effect of inactivating the condensin II complex (Csankovszki et al., 2009Csankovszki G. Collette K. Spahl K. Carey J. Snyder M. Petty E. Patel U. Tabuchi T. Liu H. McLeod I. et al.Three distinct condensin complexes control C. elegans chromosome dynamics.Curr. Biol. 2009; 19: 9-19Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) (Figure 2B, smc-4; Movie S3; see below). We observed that nuclear accumulation of GFP-AIR-2 (the C. elegans Aurora B homolog) and the commencement of chromosome condensation occur at the same time in wild-type embryos (Figure S1A). We thus asked if those two events are linked. GFP-AIR-2 nuclear accumulation occurred upon depletion of replication genes (Figures S1A and S1B, red arrows). Thus, assuming that GFP-AIR-2 and phospho-H3S10 staining serve as prophase makers, our data show that the condensation defect associated with the depletion of replication genes is not due to a lack of prophase (Figure S1A; Figure 2C). We next investigated if S phase checkpoint activation is linked to chromosome-condensation defects. As in other organisms, the inhibition of replication fork elongation in C. elegans embryos leads to the activation of a cell-cycle checkpoint delaying entry into mitosis (Brauchle et al., 2003Brauchle M. Baumer K. Gönczy P. Differential activation of the DNA replication checkpoint contributes to asynchrony of cell division in C. elegans embryos.Curr. Biol. 2003; 13: 819-827Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, Encalada et al., 2000Encalada S.E. Martin P.R. Phillips J.B. Lyczak R. Hamill D.R. Swan K.A. Bowerman B. DNA replication defects delay cell division and disrupt cell polarity in early Caenorhabditis elegans embryos.Dev. Biol. 2000; 228: 225-238Crossref PubMed Scopus (104) Google Scholar, Korzelius et al., 2011Korzelius J. The I. Ruijtenberg S. Portegijs V. Xu H. Horvitz H.R. van den Heuvel S. C. elegans MCM-4 is a general DNA replication and checkpoint component with an epidermis-specific requirement for growth and viability.Dev. Biol. 2011; 350: 358-369Crossref PubMed Scopus (23) Google Scholar). Consistent with earlier reports, inactivation of pcn-1 and rnr-1 caused a delay in nuclear envelope breakdown (Figure 2I) and rnr-1 inactivation also caused a delay in the nuclear localization of GFP-AIR-2 (Figures S1A and S1C). In contrast, cdt-1, mcm-7, and cdc45 RNAi embryos revealed little or no delay in cell-cycle progression and GFP-AIR-2 nuclear entry (Figure 2I; Figure S1C; Movies S3). These findings are consistent with a lack of replication fork initiation to such an extent that the replication checkpoint, which requires the generation of RPA-coated single-stranded DNA, cannot be activated (Zou and Elledge, 2003Zou L. Elledge S.J. Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes.Science. 2003; 300: 1542-1548Crossref PubMed Scopus (2045) Google Scholar). In line with the expected role of RPA-1 in checkpoint activation, the depletion of RPA-1 also failed to elicit a cell-cycle delay but nevertheless lead to a defect in chromosome condensation (Figures 2B, 2C, and 2I). To substantiate further this interpretation, we employed a graded depletion of MCM-7 (Figure 3A). The maximum RNAi dose (100%) led to an undetectable level of MCM-3 loaded onto anaphase chromosomes (Figures 3A and 3B) and a failure to import GFP-MCM-3 into interphase nuclei. At this 100% RNAi dose, little or no delay in nuclear envelope breakdown was observed (Figure 3C, dark bars), indicating that there was no significant activation of the replication checkpoint. Consistent with our previous results, this high RNAi dose caused a failure of chromosome condensation and the formation of anaphase bridges (Figures 3A and 3C, light bars). Lower levels of mcm-7 RNAi allowed a graded loading of GFP-MCM-3 onto chromatin (Figure 3B). RNAi doses ranging from 50% to 25% led to strong reduction of GFP-MCM-3 loading, the formation of anaphase bridges, and the maximal level of checkpoint activation as judged by lengthening of the cell cycle. This phenotype is expected if the number of chromatin-bound MCM-2–7 is insufficient to complete replication, while compromised replication triggers checkpoint activation. Limiting RNAi doses from 17% to 8% led to a less severe reduction of GFP-MCM-3 loading without checkpoint activation and without strong chromosome-condensation defects but with occasional anaphase bridges. This phenotype is consistent with the idea that an excess of MCM complexes are loaded onto “dormant” replication origins, which are not normally needed for genome duplication (Ge et al., 2007Ge X.Q. Jackson D.A. Blow J.J. Dormant origins licensed by excess Mcm2-7 are required for human cells to survive replicative stress.Genes Dev. 2007; 21: 3331-3341Crossref PubMed Scopus (415) Google Scholar, Newman et al., 2013Newman T.J. Mamun M.A. Nieduszynski C.A. Blow J.J. Replisome stall events have shaped the distribution of replication origins in the genomes of yeasts.Nucleic Acids Res. 2013; 41: 9705-9718Crossref PubMed Scopus (39) Google Scholar, Woodward et al., 2006Woodward A.M. Göhler T. Luciani M.G. Oehlmann M. Ge X. Gartner A. Jackson D.A. Blow J.J. Excess Mcm2-7 license dormant origins of replication that can be used under conditions of replicative stress.J. Cell Biol. 2006; 173: 673-683Crossref PubMed Scopus (276) Google Scholar). It is established that condensin II is required for chromosome condensation during prophase (Hirano, 2012Hirano T. Condensins: universal organizers of chromosomes with diverse functions.Genes Dev. 2012; 26: 1659-1678Crossref PubMed Scopus (245) Google Scholar, Piazza et al., 2013Piazza I. Haering C.H. Rutkowska A. Condensin: crafting the chromosome landscape.Chromosoma. 2013; 122: 175-190Crossref PubMed Scopus (29) Google Scholar, Thadani et al., 2012Thadani R. Uhlmann F. Heeger S. Condensin, chromatin crossbarring and chromosome condensation.Curr. Biol. 2012; 22: R1012-R1021Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). We therefore generated worms expressing the GFP-KLE-2 condensin II subunit. As expected from previous studies, GFP-KLE-2 was diffused throughout the nucleoplasm during S phase and concentrated on chromosomal axes during prophase, each of the chromatid axes being resolved during metaphase (Figure 2D; Movie S4). Similarly, embryos stained for another condensin II subunit, HCP-6, showed chromatin-bound HCP-6 during prophase and metaphase (Figures 2E and 2F). In contrast, inactivation of mcm-7, cdc-45, or rpa-1 prevented KLE-2 or HCP-6 foci formation during prophase (Figures 2D and 2E; Movie S4). Controls showed that KLE-2 nuclear localization required HCP-6 and that KLE-2 chromatin loading was dependent on the SMC-4 and HCP-6 condensin subunits (Figure S1D), while KLE-2 and HCP-6 were chromatin bound during mitosis when replication was blocked (Figures 2D and 2F; Movie S4). Taken together, our results suggest that condensin II concentration on chromosomal axes and chromosome condensation are both dependent on DNA having been replicated. Conversely, we found that blocking chromosome condensation by inactivating smc-4 did not inhibit chromosome duplication, as measured by EdU incorporation (Figures 2A and 2G), GFP-CDC-45 binding to chromatin (data not shown), or duplication of chromatin-bound histones (Figure 2H). Having shown that DNA replication is required for chromosome condensation during prophase, we next wanted to address the converse question, namely, whether DNA replication is required to decondense the chromatin on exit from M phase. In cell types with a significant G1 phase, chromosome decondensation occurs well before DNA replication, but in certain embryonic cells (such as Xenopus, Drosophila, and C. elegans early embryos), which have either short G1 phases or lack them entirely, it is possible that chromosome decondensation and the initiation of DNA replication occur at the same time. In order to examine precisely when S phase starts relative to chromosome decondensation, we used EdU incorporation to label C. elegans embryos during early stages of S phase in the first cell cycle after fertilization. EdU staining in the female haploid nucleus was first observed in embryos with partially condensed DNA, indicating that some DNA replication occurs during decondensation (Figure 4A, left panel red arrow, right panels for magnificati" @default.
• W1569124576 created "2016-06-24" @default.
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• W1569124576 date "2015-07-01" @default.
• W1569124576 modified "2023-10-17" @default.
• W1569124576 title "Both Chromosome Decondensation and Condensation Are Dependent on DNA Replication in C. elegans Embryos" @default.
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