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Disruption at Holliday junction Exchange of sister chromatids to form four-stranded Holliday junctions occurs naturally during meiosis, to hold sister chromatids together, and during various repair events. In eukaryotes, double Holliday junctions that escape dissolution by a helicase–topoisomerase (BTR) complex are instead processed by one of several nucleases, known as resolvases. In this study, Stephen West and colleagues define the activities of the GEN1, MUS81–EME1 and SLX1–SLX4 resolvases in the absence of BLM, the helicase component of BTR that is mutated in Bloom's syndrome. The use of these alternatives may come at a price, however, because Bloom's syndrome cells exhibit genomic instability and patients experience a broad spectrum of early-onset cancers. Exchange of sister chromatids to form four-stranded Holliday junctions occurs naturally during meiosis, to hold sister chromatids together, and during various repair events. In eukaryotes, double Holliday junctions that escape dissolution by a helicase/topoisomerase (BTR) complex are instead processed by one of several nucleases known as resolvases. This study defines the activities of the GEN1, MUS81-EME1 and SLX1-SLX4 resolvases in the absence of BLM, the helicase component of BTR that is mutated in Bloom's syndrome. In somatic cells, Holliday junctions can be formed between sister chromatids during the recombinational repair of DNA breaks or after replication fork demise. A variety of processes act upon Holliday junctions to remove them from DNA, in events that are critical for proper chromosome segregation. In human cells, the BLM protein, inactivated in individuals with Bloom’s syndrome, acts in combination with topoisomerase IIIα, RMI1 and RMI2 (BTR complex) to promote the dissolution of double Holliday junctions 1 , 2 . Cells defective for BLM exhibit elevated levels of sister chromatid exchanges (SCEs) and patients with Bloom’s syndrome develop a broad spectrum of early-onset cancers caused by chromosome instability 3 . MUS81–EME1 (refs 4–7 ), SLX1–SLX4 (refs 8–11 ) and GEN1 (refs 12 , 13 ) also process Holliday junctions but, in contrast to the BTR complex, do so by endonucleolytic cleavage. Here we deplete these nucleases from Bloom’s syndrome cells to analyse human cells compromised for the known Holliday junction dissolution/resolution pathways. We show that depletion of MUS81 and GEN1, or SLX4 and GEN1, from Bloom’s syndrome cells results in severe chromosome abnormalities, such that sister chromatids remain interlinked in a side-by-side arrangement and the chromosomes are elongated and segmented. Our results indicate that normally replicating human cells require Holliday junction processing activities to prevent sister chromatid entanglements and thereby ensure accurate chromosome condensation. This phenotype was not apparent when both MUS81 and SLX4 were depleted from Bloom’s syndrome cells, suggesting that GEN1 can compensate for their absence. Additionally, we show that depletion of MUS81 or SLX4 reduces the high frequency of SCEs in Bloom’s syndrome cells, indicating that MUS81 and SLX4 promote SCE formation, in events that may ultimately drive the chromosome instabilities that underpin early-onset cancers associated with Bloom’s syndrome.

Crosses between Drosophila melanogaster females and Drosophila simulans males produce hybrid sons that die at the larval stage. This hybrid lethality is suppressed by loss-of-function mutations in the D. melanogaster Hybrid male rescue (Hmr) or in the D. simulans Lethal hybrid rescue (Lhr) genes. Previous studies have shown that Hmr and Lhr interact with heterochromatin proteins and suppress expression of transposable elements within D. melanogaster. It also has been proposed that Hmr and Lhr function at the centromere. We examined mitotic divisions in larval brains from Hmr and Lhr single mutants and Hmr; Lhr double mutants in D. melanogaster. In none of the mutants did we observe defects in metaphase chromosome alignment or hyperploid cells, which are hallmarks of centromere or kinetochore dysfunction. In addition, we found that Hmr-HA and Lhr-HA do not colocalize with centromeres either during interphase or mitotic division. However, all mutants displayed anaphase bridges and chromosome aberrations resulting from the breakage of these bridges, predominantly at the euchromatin–heterochromatin junction. The few dividing cells present in hybrid males showed fuzzy and irregularly condensed chromosomes with unresolved sister chromatids. Despite this defect in condensation, chromosomes in hybrids managed to align on the metaphase plate and undergo anaphase. We conclude that there is no evidence for a centromeric function of Hmr and Lhr within D. melanogaster nor for a centromere defect causing hybrid lethality. Instead, we find that Hmr and Lhr are required in D. melanogaster for detachment of sister chromatids during anaphase.

Chromosome missegregation acts as one of the driving forces for chromosome instability and cancer development. Here, we find that in human cancer cells, HeLa and U2OS, depletion of 53BP1 (p53-binding protein 1) exacerbates chromosome non-disjunction resulting from a new type of sister-chromatid intertwinement, which is distinct from FANCD2-associated ultrafine DNA bridges (UFBs) induced by replication stress. Importantly, the sister DNA intertwinements trigger gross chromosomal rearrangements through a distinct process, named sister-chromatid rupture and bridging. In contrast to conventional anaphase bridge-breakage models, we demonstrate that chromatid axes of the intertwined sister-chromatids rupture prior to the breakage of the DNA bridges. Consequently, the ruptured sister arms remain tethered and cause signature chromosome rearrangements, including whole-arm (Robertsonian-like) translocation/deletion and isochromosome formation. Therefore, our study reveals a hitherto unreported chromatid damage phenomenon mediated by sister DNA intertwinements that may help to explain the development of complex karyotypes in tumour cells. Chromosome instability is associated with cancer formation. Here the authors identify in cultured human cancer cells a non-canonical DNA bridge breakage pathway leading to chromosome missegregation and rearrangements triggered by sister DNA intertwinements, which are limited by 53BP1.

DNA repair intermediates In meiotic cells, it is well established that the paired homologues are joined by a set of crossovers known as a double Holliday junction (DHJ). Whether DHJs form during mitotic recombination has been unclear, since mitotic cells possess alternative repair pathways that would not require DHJ formation. Bzymek et al . now demonstrate that mitotic and meiotic cells form similar DHJs, but that the levels in mitotic cells are approximately 10-fold lower, and show a preference for joints between sister chromatids rather than homologues. Consequently in mitotic cells non-crossover outcomes are favoured. In meiotic cells paired homologues are joined by a set of crossovers known as a double Holliday junction (DHJ). Whether DHJs form during mitotic recombination has been unclear, as mitotic cells possess alternative repair pathways that would not require DHJ formation. Here it is demonstrated that mitotic and meiotic cells form similar DHJs, but that the levels in mitotic cells are approximately 10–fold lower, and show a preference for joints between sister chromatids rather than homologues. Consequently, in mitotic cells non–crossover outcomes are favoured. Repair of DNA double-strand breaks (DSBs) by homologous recombination is crucial for cell proliferation and tumour suppression. However, despite its importance, the molecular intermediates of mitotic DSB repair remain undefined. The double Holliday junction (DHJ), presupposed to be the central intermediate for more than 25 years 1 , has only been identified during meiotic recombination 2 . Moreover, evidence has accumulated for alternative, DHJ-independent mechanisms 3 , 4 , 5 , 6 , raising the possibility that DHJs are not formed during DSB repair in mitotically cycling cells. Here we identify intermediates of DSB repair by using a budding-yeast assay system designed to mimic physiological DSB repair. This system uses diploid cells and provides the possibility for allelic recombination either between sister chromatids or between homologues, as well as direct comparison with meiotic recombination at the same locus. In mitotically cycling cells, we detect inter-homologue joint molecule (JM) intermediates whose strand composition and size are identical to those of the canonical DHJ structures observed in meiosis 2 . However, in contrast to meiosis, JMs between sister chromatids form in preference to those between homologues. Moreover, JMs seem to represent a minor pathway of DSB repair in mitotic cells, being detected at about tenfold lower levels (per DSB) than during meiotic recombination. Thus, although DHJs are identified as intermediates of DSB-promoted recombination in both mitotic and meiotic cells, their formation is distinctly regulated according to the specific dictates of the two cellular programs.

Cohesion between sister chromatids, mediated by the chromosomal cohesin complex, is a prerequisite for their alignment on the spindle apparatus and segregation in mitosis. Budding yeast cohesin first associates with chromosomes in G1. Then, during DNA replication in S-phase, the replication fork-associated acetyltransferase Eco1 acetylates the cohesin subunit Smc3 to make cohesin’s DNA binding resistant to destabilization by the Wapl protein. Whether stabilization of cohesin molecules that happen to link sister chromatids is sufficient to build sister chromatid cohesion, or whether additional reactions are required to establish these links, is not known. In addition to Eco1, several other factors contribute to cohesion establishment, including Ctf4, Ctf18, Tof1, Csm3, Chl1 and Mrc1, but little is known about their roles. Here, we show that each of these factors facilitates cohesin acetylation. Moreover, the absence of Ctf4 and Chl1, but not of the other factors, causes a synthetic growth defect in cells lacking Eco1. Distinct from acetylation defects, sister chromatid cohesion in ctf4Δ and chl1Δ cells is not improved by removing Wapl. Unlike previously thought, we do not find evidence for a role of Ctf4 and Chl1 in Okazaki fragment processing, or of Okazaki fragment processing in sister chromatid cohesion. Thus, Ctf4 and Chl1 delineate an additional acetylation-independent pathway that might hold important clues as to the mechanism of sister chromatid cohesion establishment.

Cohesin is essential for sister chromatid cohesion, which ensures equal segregation of the chromatids to daughter cells. However, the molecular mechanism by which cohesin mediates this function is elusive. Scc3, one of the four core subunits of cohesin, is vital to cohesin activity. However, the mechanism by which Scc3 contributes to the activity and identity of its functional domains is not fully understood. Here, we describe an in-frame five-amino acid insertion mutation after glutamic acid 704 (scc3-E704ins) in yeast Scc3, located in the middle of the second armadillo repeat. Mutated cohesin-scc3-E704ins complexes are unable to establish cohesion. Detailed molecular and genetic analyses revealed that the mutated cohesin has reduced affinity to the Scc2 loader. This inhibits its enrichment at centromeres and chromosomal arms. Mutant complexes show a slow diffusion rate in live cells suggesting that they induce a major conformational change in the complex. The analysis of systematic mutations in the insertion region of Scc3 revealed two conserved aspartic acid residues that are essential for the activity. The study offers a better understanding of the contribution of Scc3 to cohesin activity and the mechanism by which cohesin tethers the sister chromatids during the cell cycle.

Catenation links between sister chromatids are formed progressively during DNA replication and are involved in the establishment of sister chromatid cohesion. Topo IV is a bacterial type II topoisomerase involved in the removal of catenation links both behind replication forks and after replication during the final separation of sister chromosomes. We have investigated the global DNA-binding and catalytic activity of Topo IV in E. coli using genomic and molecular biology approaches. ChIP-seq revealed that Topo IV interaction with the E. coli chromosome is controlled by DNA replication. During replication, Topo IV has access to most of the genome but only selects a few hundred specific sites for its activity. Local chromatin and gene expression context influence site selection. Moreover strong DNA-binding and catalytic activities are found at the chromosome dimer resolution site, dif, located opposite the origin of replication. We reveal a physical and functional interaction between Topo IV and the XerCD recombinases acting at the dif site. This interaction is modulated by MatP, a protein involved in the organization of the Ter macrodomain. These results show that Topo IV, XerCD/dif and MatP are part of a network dedicated to the final step of chromosome management during the cell cycle.

The correct separation of homologous chromosomes during meiosis I, and sister chromatids during meiosis II, relies on the tight control of the cohesion complex. The phosphorylation and subsequent cleavage of the meiotic recombination protein REC8 (REC8-like family protein [SYN1] in Arabidopsis [ ]), the α-kleisin subunit of the cohesion ring, along the chromosome arms at meiosis I allows crossovers and separation of homologous chromosomes without chromatid dissociation. REC8 continues to localize and function at the centromeres up to metaphase II and, in yeast and vertebrates, is protected from cleavage by means of protein phosphatase 2A (PP2A)-mediated dephosphorylation. Here, we show that, in plants, centromeric sister chromatid cohesion until meiosis II also requires the activity of a PP2A-type phosphatase complex. The combined absence of the regulatory subunits PP2AB'α and PP2AB'β leads to the premature loss of chromosome cohesion in meiosis I. Male meiocytes of the double mutant display premature depletion of SYN1. The PP2AA1 structural and B'α regulatory subunit localize specifically to centromeres until metaphase II, supporting a role for the PP2A complex in the SYN1-mediated maintenance of centromeric cohesion in plant meiosis.

Genotoxic DNA double-strand breaks (DSBs) can be repaired by error-free homologous recombination (HR) or mutagenic non-homologous end-joining 1 . HR supresses tumorigenesis 1 , but is restricted to the S and G2 phases of the cell cycle when a sister chromatid is present 2 . Breast cancer type 1 susceptibility protein (BRCA1) promotes HR by antagonizing the anti-resection factor TP53-binding protein 1(53BP1) (refs. 2 , 3 , 4 – 5 ), but it remains unknown how BRCA1 function is limited to the S and G2 phases. We show that BRCA1 recruitment requires recognition of histone H4 unmethylated at lysine 20 (H4K20me0), linking DSB repair pathway choice directly to sister chromatid availability. We identify the ankyrin repeat domain of BRCA1-associated RING domain protein 1 (BARD1)—the obligate BRCA1 binding partner 3 —as a reader of H4K20me0 present on new histones in post-replicative chromatin 6 . BARD1 ankyrin repeat domain mutations disabling H4K20me0 recognition abrogate accumulation of BRCA1 at DSBs, causing aberrant build-up of 53BP1, and allowing anti-resection activity to prevail in S and G2. Consequently, BARD1 recognition of H4K20me0 is required for HR and resistance to poly (ADP-ribose) polymerase inhibitors. Collectively, this reveals that BRCA1–BARD1 monitors the replicative state of the genome to oppose 53BP1 function, routing only DSBs within sister chromatids to HR. Nakamura et al. show that DNA repair pathway choice and initiation of homologous recombination is guided by the recruitment of BRCA1 to post-replicative chromatin by BARD1 recognition of histone H4 tails unmethylated on lysine 20.

Mitosis and meiosis are two distinct cell division programs. During mitosis, sister chromatids separate, whereas during the first meiotic division, homologous chromosomes pair and then segregate from each other. In most organisms, germ cells do both programs sequentially, as they first amplify through mitosis, before switching to meiosis to produce haploid gametes. Here, we show that autosomal chromosomes are unpaired at their centromeres in Drosophila germline stem cells, and become paired during the following four mitosis of the differentiating daughter cell. Surprisingly, we further demonstrate that components of the central region of the synaptonemal complex are already expressed in the mitotic region of the ovaries, localize close to centromeres, and promote de novo association of centromeres. Our results thus show that meiotic proteins and meiotic organization of centromeres, which are key features to ensure reductional segregation, are laid out in amplifying germ cells, before meiosis has started.