Physical Proximity of Sister Chromatids Promotes Top2-Dependent Intertwining.

Sister chromatid intertwines (SCIs), or catenanes, are topological links between replicated chromatids that interfere with chromosome segregation. The formation of SCIs is thought to be a consequence of fork swiveling during DNA replication, and their removal is thought to occur because of the intrinsic feature of type II topoisomerases (Top2) to simplify DNA topology. Here, we report that SCIs are also formed independently of DNA replication during G2/M by Top2-dependent concatenation of cohesed chromatids due to their physical proximity. We demonstrate that, in contrast to G2/M, Top2 removes SCIs from cohesed chromatids at the anaphase onset. Importantly, SCI removal in anaphase requires condensin and coincides with the hyperactivation of condensin DNA supercoiling activity. This is consistent with the longstanding proposal that condensin provides a bias in Top2 function toward decatenation. A comprehensive model for the formation and resolution of toxic SCI entanglements on eukaryotic genomes is proposed.

Condensin Relocalization from Centromeres to Chromosome Arms Promotes Top2 Recruitment during Anaphase.

Condensin is a conserved chromosomal complex necessary to promote mitotic chromosome condensation and sister chromatid resolution during anaphase. Here, we report that yeast condensin binds to replicated centromere regions. We show that centromeric condensin relocalizes to chromosome arms as cells undergo anaphase segregation. We find that condensin relocalization is initiated immediately after the bipolar attachment of sister kinetochores to spindles and requires Polo kinase activity. Moreover, condensin localization during anaphase involves a higher binding rate on DNA and temporally overlaps with condensin's DNA overwinding activity. Finally, we demonstrate that topoisomerase 2 (Top2) is also recruited to chromosome arms during anaphase in a condensin-dependent manner. Our results uncover a functional relation between condensin and Top2 during anaphase to mediate chromosome segregation.

Post-replicative repair involves separase-dependent removal of the kleisin subunit of cohesin.

DNA double-strand break repair is critical for cell viability and involves highly coordinated pathways to restore DNA integrity at the lesion. An early event during homology-dependent repair is resection of the break to generate progressively longer 3' single-strand tails that are used to identify suitable templates for repair. Sister chromatids provide near-perfect sequence homology and are therefore the preferred templates during homologous recombination. To provide a bias for the use of sisters as donors, cohesin--the complex that tethers sister chromatids together--is recruited to the break to enforce physical proximity. Here we show that DNA breaks promote dissociation of cohesin loaded during the previous S phase in budding yeast, and that damage-induced dissociation of cohesin requires separase, the protease that dissolves cohesion in anaphase. Moreover, a separase-resistant allele of the gene coding for the α-kleisin subunit of cohesin, Mcd1 (also known as Scc1), reduces double-strand break resection and compromises the efficiency of repair even when loaded during DNA damage. We conclude that post-replicative DNA repair involves cohesin dissociation by separase to promote accessibility to repair factors during the coordinated cellular response to restore DNA integrity.

SUMOylation of the α-kleisin subunit of cohesin is required for DNA damage-induced cohesion.

BACKGROUND: Cohesion between sister chromatids is fundamental to ensure faithful chromosome segregation during mitosis and accurate repair of DNA damage postreplication. At the molecular level, cohesion establishment involves two defined events, a chromatin binding step and a chromatid entrapment event driven by posttranslational modifications on cohesin subunits. RESULTS: Here, we show that modification by the small ubiquitin-like protein (SUMO) is required for sister chromatid tethering after DNA damage. We find that all subunits of cohesin become SUMOylated upon exposure to DNA damaging agents or presence of a DNA double-strand break. We have mapped all lysine residues on cohesin's α-kleisin subunit Mcd1 (Scc1) where SUMO can conjugate. We demonstrate that Mcd1 SUMOylation-deficient alleles are still recruited to DSB-proximal regions but are defective in tethering sister chromatids and consequently fail to establish damage-induced cohesion both at DSBs and undamaged chromosomes. Moreover, we demonstrate that the bulk of Mcd1 SUMOylation in response to damage is carried out by the SUMO E3 ligase Nse2, a subunit of the related Smc5-Smc6 complex. SUMOylation occurs in cells with compromised Chk1 kinase activity, necessary for known posttranslational modifications on Mcd1, required for damage-induced cohesion. CONCLUSIONS: These findings demonstrate that SUMOylation of Mcd1 is a novel prerequisite for the establishment of DNA damage-induced cohesion at DSB-proximal regions and cohesion-associating regions (CARs) genome-wide.

Cdc14 phosphatase promotes segregation of telomeres through repression of RNA polymerase II transcription.

Kinases and phosphatases regulate messenger RNA synthesis through post-translational modification of the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (ref. 1). In yeast, the phosphatase Cdc14 is required for mitotic exit(2,3) and for segregation of repetitive regions(4). Cdc14 is also a subunit of the silencing complex RENT (refs 5,6), but no roles in transcriptional repression have been described. Here we report that inactivation of Cdc14 causes silencing defects at the intergenic spacer sequences of ribosomal genes during interphase and at Y' repeats in subtelomeric regions during mitosis. We show that the role of Cdc14 in silencing is independent of the RENT deacetylase subunit Sir2. Instead, Cdc14 acts directly on RNA polymerase II by targeting CTD phosphorylation at Ser 2 and Ser 5. We also find that the role of Cdc14 as a CTD phosphatase is conserved in humans. Finally, telomere segregation defects in cdc14 mutants(4) correlate with the presence of subtelomeric Y' elements and can be rescued by transcriptional inhibition of RNA polymerase II.