Here we report the ®rst characterization of replication timing and its regulation in the ®ssion yeast Schizosaccharomyces pombe. We used three different synchronization methods: centrifugal elutriation, cdc10 temperature-shift and release, and starvation for deoxyribonucleoside triphosphates (dNTPs) by treatment with hydroxyurea (HU) followed by removal of HU, to study the times when speci®c autonomously replicating sequence elements (ARS elements; potential replication origins) replicate during S phase. We found that individual ARS elements replicate at characteristic times, some early and some late, independently of synchronization method. In wild-type cells treated with HU, early ARS elements replicated but late ones did not. However, in HU-treated mutant cells lacking the Rad3 (similar to human ATR and ATM) or Cds1 (similar to human CHK2) checkpoint kinase, both early and late ARS elements were able to replicate. Thus under conditions of dNTP starvation the Rad3 and Cds1 kinases are needed to suppress the replication of normally late-replicating regions.

Claspin is necessary for the ATR-dependent activation of Chk1 in

Euchromatin, which has an open structure and is frequently transcribed, tends to replicate in early S phase. Heterochromatin, which is more condensed and rarely transcribed, usually replicates in late S phase. Here, we report significant deviation from this correlation in the fission yeast, Schizosaccharomyces pombe. We found that heterochromatic centromeres and silent matingtype cassettes replicate in early S phase. Only heterochromatic telomeres replicate in late S phase. Research in other laboratories has shown that occasionally other organisms also replicate some of their heterochromatin in early S phase. Thus, late replication is not an obligatory feature of heterochromatin. "Heterochromatin" was originally defined as chromatin that remains condensed during interphase, whereas "euchromatin" decondenses during interphase (Heitz 1928(Heitz , 1929. Later, the discovery that genes from euchromatin can become epigenetically inactivated if they are translocated into heterochromatin (for review, see Grewal and Elgin 2002) provided an additional, functional definition for heterochromatin. Recent studies have begun to provide a molecular definition as well (for review, see Grewal and Elgin 2002). Many forms of heterochromatin are characterized by hypermethylation of Lys 9 on histone H3 (H3-K9). In these cases, a protein with a chromodomain and a chromo-shadow domain (similar to Drosophila Hp1 and fission yeast Swi6) binds to methylated H3-K9 with its chromodomain, and then recruits additional proteins to the heterochromatic region with its chromo-shadow domain.Until now, replication in late S phase has been considered to be another distinguishing feature of heterochromatin. The paradigm of late heterochromatin replication was first articulated by Lima-de-Faria and Jaworska (1968) on the basis of studies in a wide range of eukaryotic organisms. With minor exceptions, this paradigm has withstood the test of time (for review, see Gilbert 2002). Now, however, we report significant deviation from this paradigm in fission yeast. Results and Discussion Previous results: telomeresChromatin near fission yeast telomeres is heterochromatic, because bringing normally active genes into the proximity of telomeres epigenetically inactivates the genes, and this inactivation is partially dependent on standard heterochromatin proteins including Swi6 (Nimmo et al. 1994;Allshire et al. 1995;Ekwall et al. 1995Ekwall et al. , 1996. Epigenetic inactivation of euchromatic genes introduced into telomere-proximal positions is called Telomere Position Effect (TPE) and is conserved from budding yeast (Gottschling et al. 1990;Tham and Zakian 2002) to humans (Baur et al. 2001).Using several independent synchronization procedures, we have demonstrated previously that the terminal HindIII restriction fragments of fission yeast chromosomes I and II replicate in very late S phase (Kim and Huberman 2001), consistent with the paradigm of late heterochromatin replication. However, we were surprised when the same studies revealed that the outer portions...