Eukaryotic chromosomal DNA replication requires a two-step assembly of replication proteins on origins; formation of the prereplicative complex (pre-RC) in late M and G1 phases of the cell cycle, and assembly of other replication proteins in S phase to load DNA polymerases to initiate DNA synthesis. In budding yeast, assembly of Dpb11 and the Sld3-Cdc45 complex on the pre-RC at origins is required for loading DNA polymerases. Here we describe a novel replication complex, GINS (Go, Ichi, Nii, and San; five, one, two, and three in Japanese), in budding yeast, consisting of Sld5, Psf1 (partner of Sld five 1), Psf2, and Psf3 proteins, all of which are highly conserved in eukaryotic cells. Since the conditional mutations of Sld5 and Psf1 confer defect of DNA replication under nonpermissive conditions, GINS is suggested to function for chromosomal DNA replication. Consistently, in S phase, GINS associates first with replication origins and then with neighboring sequences. Without GINS, neither Dpb11 nor Cdc45 associates properly with chromatin DNA. Conversely, without Dpb11 or Sld3, GINS does not associate with origins. Moreover, genetic and two-hybrid interactions suggest that GINS interacts with Sld3 and Dpb11. Therefore, Dpb11, Sld3, Cdc45, and GINS assemble in a mutually dependent manner on replication origins to initiate DNA synthesis.[Keywords: DNA replication; replication protein; Dpb11; Sld3; Sld5] Supplemental material is available at http://www.genesdev.org.

CENP-A is a centromere-specific histone H3 variant that is essential for kinetochore formation. Here, we report that the fission yeast Schizosaccharomyces pombe has at least two distinct CENP-A deposition phases across the cell cycle: S and G2. The S phase deposition requires Ams2 GATA factor, which promotes histone gene activation. In ⌬ams2, CENP-A fails to retain during S, but it reaccumulates onto centromeres via the G2 deposition pathway, which is down-regulated by Hip1, a homologue of HIRA histone chaperon. Reducing the length of G2 in ⌬ams2 results in failure of CENP-A accumulation, leading to chromosome missegregation. N-terminal green fluorescent protein-tagging reduces the centromeric association of CENP-A, causing cell death in ⌬ams2 but not in wild-type cells, suggesting that the N-terminal tail of CENP-A may play a pivotal role in the formation of centromeric nucleosomes at G2. These observations imply that CENP-A is normally localized to centromeres in S phase in an Ams2-dependent manner and that the G2 pathway may salvage CENP-A assembly to promote genome stability. The flexibility of CENP-A incorporation during the cell cycle may account for the plasticity of kinetochore formation when the authentic centromere is damaged. INTRODUCTIONThe kinetochore is a multiprotein-DNA complex that is indispensable for chromosome segregation and that normally forms on a single chromosomal locus, the centromere (Cleveland et al., 2003). Lack of a kinetochore or formation of multiple kinetochores on a chromosome may have deleterious effects on mitosis (Karpen and Allshire, 1997;Amor and Choo, 2002;Henikoff and Dalal, 2005). CENP-A represents the most likely candidate for the epigenetic "mark" responsible for the maintenance of centromere identity (Black et al., 2004. Because reformation of CENP-A-containing nucleosomes after DNA synthesis is thought to be a prerequisite for mitotic kinetochore assembly, precise targeting of CENP-A into a single, restricted locus on each chromosome before cell division is essential for cell viability . At least three components that affect CENP-A localization, the Mis16 -Mis18 complex (Hayashi et al., 2004;Fujita et al., 2007), the Mis6 -Sim4 complex (Takahashi et al., 2000;Pidoux et al., 2003), and Ams2 GATA-type transcription factor (Chen et al., 2003a), have been identified in the fission yeast Schizosaccharomyces pombe, which is an ideal model organism in which to study complex centromere structure and function (Takahashi et al., 1992;Karpen and Allshire, 1997).Which phase of the cell cycle is used for CENP-A incorporation remains controversial. During S phase, canonical core histones have been suggested to be deposited into duplicated DNAs in a semiconservative manner (Tagami et al., 2004;Natsume et al., 2007). Experiments using fluorescence recovery after photobleaching demonstrated that CENP-A of the budding yeast Saccharomyces cerevisiae is recruited to centromeres coincident with DNA synthesis (Pearson et al., 2004), presumably reflecting disassembly and reassembly of cent...

The histone genes are highly reiterated in a wide range of eukaryotic genomes. The fission yeast, Schizosaccharomyces pombe, has three pairs of histone H3-H4 genes: hht1+-hhf1+, hht2+-hhf2+ and hht3+-hhf3+. While the deduced amino acid sequences are identical, it remains unknown whether transcriptional regulation differs among the three pairs. Here, we report the transcriptional properties of each H3-H4 gene pair during the cell cycle. The levels of transcripts of hht1+-hhf1+ and hht3+-hhf3+ pairs and hhf2+ are increased at S-phase, while that of hht2+ remains constant throughout the cell cycle. We showed that the GATA-type transcription factor, Ams2, binds to the promoter regions of core histone genes in an AACCCT-box-dependent manner and is required for activation of S-phase-specific transcription. Furthermore, we found that Ams2-depletion stimulates feedback regulation of histone transcripts, mainly up-regulating the basal levels of hht2+-hhf2+ transcription, which are normally down-regulated by Hip1 and Slm9, homologs of the human histone chaperone, HIRA. These observations provide insight into the molecular mechanisms of differential regulation of transcripts from repeated histone genes in the fission yeast.

Chromosomes with multiple centromeres are stabilized by epigenetic centromere inactivation, which is initiated by kinetochore disassembly. Consequent heterochromatinization and histone deacetylation expanding from pericentric repeats to the central domain prevent reactivation of the inactivated centromere.

CENP-A is a centromere-specific histone H3 variant that is-essential for faithful chromosome segregation in all eukaryotes thus far investigated. We genetically identified two factors, Ams2 and Mis6, each of which is required for the correct centromere localization of SpCENP-A (Cnp1), the fission yeast homologue of CENP-A. Ams2 is a cell-cycle-regulated GATA factor that localizes on the nuclear chromatin, including on centromeres, during the S phase. Ams2 may be responsible for the replication-coupled loading of SpCENP-A by facilitating nucleosomal formation during the S phase. Consistently, overproduction of histone H4, but not that of H3, suppressed the defect of SpCENP-A localization in Ams2-deficient cells. We demonstrated the existence of at least two distinct phases for SpCENP-A loading during the cell cycle: the S phase and the late-G2 phase. Ectopically induced SpCENP-A was efficiently loaded onto the centromeres in G2-arrested cells, indicating that SpCENP-A probably undergoes replication-uncoupled loading after the completion of S phase. This G2 loading pathway of SpCENP-A may require Mis6, a constitutive centromere-binding protein that is also implicated in the Mad2-dependent spindle attachment checkpoint response. Here, we discuss the functional relationship between the flexible loading mechanism of CENP-A and the plasticity of centromere chromatin formation in fission yeast.