The checkpoint regulatory mechanism has an important role in maintaining the integrity of the genome. This is particularly important in S phase of the cell cycle, when genomic DNA is most susceptible to various environmental hazards. When chemical agents damage DNA, activation of checkpoint signalling pathways results in a temporary cessation of DNA replication. A replication-pausing complex is believed to be created at the arrested forks to activate further checkpoint cascades, leading to repair of the damaged DNA. Thus, checkpoint factors are thought to act not only to arrest replication but also to maintain a stable replication complex at replication forks. However, the molecular mechanism coupling checkpoint regulation and replication arrest is unknown. Here we demonstrate that the checkpoint regulatory proteins Tof1 and Mrc1 interact directly with the DNA replication machinery in Saccharomyces cerevisiae. When hydroxyurea blocks chromosomal replication, this assembly forms a stable pausing structure that serves to anchor subsequent DNA repair events.

Sister chromatids, the products of eukaryotic DNA replication, are held together after their synthesis by the chromosomal cohesin complex. This allows the spindle in mitosis to recognise pairs of replication products for segregation into opposite direction1-6. Cohesin forms large protein rings that may bind DNA strands by encircling7, but the characterisation of cohesin binding to chromosomes in vivo has remained vague. Here, we present high resolution analysis of cohesin association along budding yeast chromosomes III -VI. Cohesin localises almost exclusively between genes transcribed in converging direction. We find that not the underlying sequence, but active transcription positions cohesin at these sites. Cohesin is initially loaded onto chromosomes at separate places, marked by the Scc2/Scc4 cohesin loading complex8, from where it appears to slide to its more permanent locations. But even after sister chromatid cohesion is established changes in transcription lead to repositioning of cohesin. Thus a key architectural feature of mitotic chromosomes, the sites of cohesin binding and therefore most likely sister chromatid cohesion, display surprising flexibility. Cohesin localisation to places of convergent transcription is conserved in fission yeast, suggesting that it is a common feature of eukaryotic chromosomes.Correspondence and requests for materials should be addressed to F.U. (e-mail: frank.uhlmann@cancer.org.uk).. * these authors contributed equally Supplementary Information accompanies the paper on Nature's website (http://www.nature.com). Cohesin association with yeast and human chromosomes has been studied4,9-15, but the defining characteristics of association sites, and how cohesin gets to these sites, remained unclear. We analysed cohesin binding to chromosome VI of the budding yeast Saccharomyces cerevisiae by chromatin immunoprecipitation (ChIP) followed by hybridisation to a high-density oligonucleotide array16. The pattern of association in metaphase was similar for all cohesin subunits analysed, Scc1, Scc3, Smc3, and Pds5 ( Fig. 1, and Supplementary Figure S1). It was also similar before the establishment of sister chromatid cohesion, in cells arrested with the replication inhibitor hydroxyurea (Ref. 9, and Supplementary Figure S1). Cohesin bound 28 distinct sites, each spanning 1-4 kilobases (kb) in width. The intensity of association varied, with the strongest peaks found around the centromere, consistent with previous analyses9-11. The distance between neighbouring cohesin association sites ranged from 2 to 35 kb. Almost all cohesin association sites were centred in intergenic regions where genes from opposite strands converged (Fig. 1a), as previously suggested17. Using an additional high-density array, we also mapped the association of Scc1 with chromosomes III, IV, and V (Supplementary Table 1 and Figure S2). 91% (276 of 304) cohesin association sites identified lie at intergenic regions between converging genes, and of 328 convergent intergene regions 84% were bound by cohes...

Cornelia de Lange syndrome (CdLS) is a dominantly inherited congenital malformation disorder caused by mutations in the cohesin-loading protein NIPBL1,2 for nearly 60% of individuals with classical CdLS3-5 and in the core cohesin components SMC1A (~5%) and SMC3 (<1%) for a smaller fraction of probands6,7. In humans, the multi-subunit complex cohesin is comprised of SMC1, SMC3, RAD21 and a STAG protein to form a ring structure proposed to encircle sister chromatids to mediate sister chromatid cohesion (SCC)8 as well as play key roles in gene regulation9. SMC3 is acetylated during S-phase to establish cohesiveness of chromatin-loaded cohesin10-13 and in yeast, HOS1, a class I histone deacetylase, deacetylates SMC3 during anaphase14-16. Here we report the identification of HDAC8 as the vertebrate SMC3 deacetylase as well as loss-of-function HDAC8 mutations in six CdLS probands. Loss of HDAC8 activity results in increased SMC3 acetylation (SMC3-ac) and inefficient dissolution of the “used” cohesin complex released from chromatin in both prophase and anaphase. While SMC3 with retained acetylation is loaded onto chromatin, ChIP-Seq analysis demonstrates decreased occupancy of cohesin localization sites that results in a consistent pattern of altered transcription seen in CdLS cell lines with either NIPBL or HDAC8 mutations.

[Keywords: Chromosome condensation; condensin; Scc2/4; tRNA genes; TFIIIC] Supplemental material is available at http://www.genesdev.org.

Segregation of homologous maternal and paternal centromeres to opposite poles during meiosis I depends on post-replicative crossing over between homologous non-sister chromatids, which creates chiasmata and therefore bivalent chromosomes. Destruction of sister chromatid cohesion along chromosome arms due to proteolytic cleavage of cohesin's Rec8 subunit by separase resolves chiasmata and thereby triggers the first meiotic division. This produces univalent chromosomes, the chromatids of which are held together by centromeric cohesin that has been protected from separase by shugoshin (Sgo1/MEI-S332) proteins. Here we show in both fission and budding yeast that Sgo1 recruits to centromeres a specific form of protein phosphatase 2A (PP2A). Its inactivation causes loss of centromeric cohesin at anaphase I and random segregation of sister centromeres at the second meiotic division. Artificial recruitment of PP2A to chromosome arms prevents Rec8 phosphorylation and hinders resolution of chiasmata. Our data are consistent with the notion that efficient cleavage of Rec8 requires phosphorylation of cohesin and that this is blocked by PP2A at meiosis I centromeres.