Imperial College London

ProfessorLuisAragon Alcaide

Faculty of MedicineInstitute of Clinical Sciences

Professor of Genetics
 
 
 
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Contact

 

+44 (0)20 3313 8013luis.aragon

 
 
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Location

 

CRB (Clinical Research Building)Hammersmith Campus

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Summary

 

Publications

Publication Type
Year
to

40 results found

Bermudez-Lopez M, Teresa Villoria M, Esteras M, Jarmuz A, Torres-Rosell J, Clemente-Blanco A, Aragon L, Bermúdez-López M, Villoria MT, Esteras M, Jarmuz A, Torres-Rosell J, Clemente-Blanco A, Aragon L, Bermúdez-López M, Villoria MT, Esteras M, Jarmuz A, Torres-Rosell J, Clemente-Blanco A, Aragon L, Bermúdez-López M, Villoria MT, Esteras M, Jarmuz A, Torres-Rosell J, Clemente-Blanco A, Aragon L, Bermudez-Lopez M, Villoria MT, Esteras M, Jarmuz A, Torres-Rosell J, Clemente-Blanco A, Aragon Alcaide Let al., 2016, Sgs1's roles in DNA end resection, HJ dissolution, and crossover suppression require a two-step SUMO regulation dependent on Smc5/6, GENES & DEVELOPMENT, Vol: 30, Pages: 1339-1356, ISSN: 0890-9369

The RecQ helicase Sgs1 plays critical roles during DNA repair by homologous recombination, from end resection to Holliday junction (HJ) dissolution. Sgs1 has both pro- and anti-recombinogenic roles, and therefore its activity must be tightly regulated. However, the controls involved in recruitment and activation of Sgs1 at damaged sites are unknown. Here we show a two-step role for Smc5/6 in recruiting and activating Sgs1 through SUMOylation. First, auto-SUMOylation of Smc5/6 subunits leads to recruitment of Sgs1 as part of the STR (Sgs1-Top3-Rmi1) complex, mediated by two SUMO-interacting motifs (SIMs) on Sgs1 that specifically recognize SUMOylated Smc5/6. Second, Smc5/6-dependent SUMOylation of Sgs1 and Top3 is required for the efficient function of STR. Sgs1 mutants impaired in recognition of SUMOylated Smc5/6 (sgs1-SIMΔ) or SUMO-dead alleles (sgs1-KR) exhibit unprocessed HJs at damaged replication forks, increased crossover frequencies during double-strand break repair, and severe impairment in DNA end resection. Smc5/6 is a key regulator of Sgs1's recombination functions.

JOURNAL ARTICLE

Sen N, Leonard J, Torres R, Garcia-Luis J, Palou-Marin G, Aragon L, Sen N, Leonard J, Torres R, Garcia-Luis J, Palou-Marin G, Aragón L, Sen N, Leonard J, Torres R, Garcia-Luis J, Palou-Marin G, Aragón L, Sen N, Leonard J, Torres R, Garcia-Luis J, Palou-Marin G, Aragón L, Sen N, Leonard J, Torres R, Garcia-Luis J, Palou-Marin G, Aragon Alcaide Let al., 2016, Physical Proximity of Sister Chromatids Promotes Top2-Dependent Intertwining, MOLECULAR CELL, Vol: 64, Pages: 134-147, ISSN: 1097-2765

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.

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Leonard J, Sen N, Torres R, Sutani T, Jarmuz A, Shirahige K, Aragon L, Leonard J, Sen N, Torres R, Sutani T, Jarmuz A, Shirahige K, Aragón L, Leonard J, Sen N, Torres R, Sutani T, Jarmuz A, Shirahige K, Aragón L, Leonard J, Sen N, Torres R, Sutani T, Jarmuz A, Shirahige K, Aragón L, Leonard J, Sen N, Torres R, Sutani T, Jarmuz A, Shirahige K, Aragon Let al., 2015, Condensin Relocalization from Centromeres to Chromosome Arms Promotes Top2 Recruitment during Anaphase, CELL REPORTS, Vol: 13, Pages: 2336-2344, ISSN: 2211-1247

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.

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Aragon L, Martinez-Perez E, Merkenschlager M, Aragon L, Martinez-Perez E, Merkenschlager M, Aragon L, Martinez-Perez E, Merkenschlager M, Aragon L, Martinez-Perez E, Merkenschlager Met al., 2013, Condensin, cohesin and the control of chromatin states, CURRENT OPINION IN GENETICS & DEVELOPMENT, Vol: 23, Pages: 204-211, ISSN: 0959-437X

Cohesin and condensin complexes are essential for defining the topology of chromosomes through the cell cycle. Here we look at the emerging role of these complexes in regulating chromatin structure and gene expression and reflect on how these activities could be linked with chromosome topology.

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McAleenan A, Clemente-Blanco A, Cordon-Preciado V, Sen N, Esteras M, Jarmuz A, Aragon L, McAleenan A, Clemente-Blanco A, Cordon-Preciado V, Sen N, Esteras M, Jarmuz A, Aragón L, McAleenan A, Clemente-Blanco A, Cordon-Preciado V, Sen N, Esteras M, Jarmuz A, Aragón L, McAleenan A, Clemente-Blanco A, Cordon-Preciado V, Sen N, Esteras M, Jarmuz A, Aragón Let al., 2013, Post-replicative repair involves separase-dependent removal of the kleisin subunit of cohesin, NATURE, Vol: 493, Pages: 250-U270, ISSN: 0028-0836

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.

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Baxter J, Aragon L, Baxter J, Aragón L, Baxter J, Aragón L, Baxter J, Aragón Let al., 2012, A model for chromosome condensation based on the interplay between condensin and topoisomerase II, TRENDS IN GENETICS, Vol: 28, Pages: 110-117, ISSN: 0168-9525

The compaction of chromatin that occurs when cells enter mitosis is probably the most iconic process of dividing cells. Mitotic chromosomal compaction or 'condensation' is functionally linked to resolution of chromosomal intertwines, transcriptional shut-off and complete segregation of chromosomes. At present, understanding of the molecular events required to convert interphase chromatin into mitotic chromosomes is limited. Here, we review recent advances in the field, focusing on potential chromosomal compaction mechanisms and their importance to chromosome segregation. We propose a model of how metaphase chromosomes could be shaped based on the enzymatic activities of condensin and topoisomerase II in overwinding and relaxation of the DNA fiber during mitosis. We suggest that condensin overwinding is an important requirement for intertwine resolution by topoisomerase II and, together with the inhibition of transcription, contributes to cytological mitotic chromosome appearance or 'condensation'.

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McAleenan A, Cordon-Preciado V, Clemente-Blanco A, Liu I-C, Sen N, Leonard J, Jarmuz A, Aragon L, McAleenan A, Cordon-Preciado V, Clemente-Blanco A, Liu IC, Sen N, Leonard J, Jarmuz A, Aragón L, McAleenan A, Cordon-Preciado V, Clemente-Blanco A, Liu I-C, Sen N, Leonard J, Jarmuz A, Aragón L, McAleenan A, Cordon-Preciado V, Clemente-Blanco A, Liu I-C, Sen N, Leonard J, Jarmuz A, Aragón Let al., 2012, SUMOylation of the alpha-Kleisin Subunit of Cohesin Is Required for DNA Damage-Induced Cohesion, CURRENT BIOLOGY, Vol: 22, Pages: 1564-1575, ISSN: 0960-9822

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.

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Quevedo O, Garcia-Luis J, Matos-Perdomo E, Aragon L, Machin F, Quevedo O, García-Luis J, Matos-Perdomo E, Aragón L, Machín F, Quevedo O, García-Luis J, Matos-Perdomo E, Aragón L, Machín F, Quevedo O, García-Luis J, Matos-Perdomo E, Aragón L, Machín Fet al., 2012, Nondisjunction of a Single Chromosome Leads to Breakage and Activation of DNA Damage Checkpoint in G2, PLOS GENETICS, Vol: 8, Pages: e1002509-e1002509, ISSN: 1553-7390

The resolution of chromosomes during anaphase is a key step in mitosis. Failure to disjoin chromatids compromises the fidelity of chromosome inheritance and generates aneuploidy and chromosome rearrangements, conditions linked to cancer development. Inactivation of topoisomerase II, condensin, or separase leads to gross chromosome nondisjunction. However, the fate of cells when one or a few chromosomes fail to separate has not been determined. Here, we describe a genetic system to induce mitotic progression in the presence of nondisjunction in yeast chromosome XII right arm (cXIIr), which allows the characterisation of the cellular fate of the progeny. Surprisingly, we find that the execution of karyokinesis and cytokinesis is timely and produces severing of cXIIr on or near the repetitive ribosomal gene array. Consequently, one end of the broken chromatid finishes up in each of the new daughter cells, generating a novel type of one-ended double-strand break. Importantly, both daughter cells enter a new cycle and the damage is not detected until the next G2, when cells arrest in a Rad9-dependent manner. Cytologically, we observed the accumulation of damage foci containing RPA/Rad52 proteins but failed to detect Mre11, indicating that cells attempt to repair both chromosome arms through a MRX-independent recombinational pathway. Finally, we analysed several surviving colonies arising after just one cell cycle with cXIIr nondisjunction. We found that aberrant forms of the chromosome were recovered, especially when RAD52 was deleted. Our results demonstrate that, in yeast cells, the Rad9-DNA damage checkpoint plays an important role responding to compromised genome integrity caused by mitotic nondisjunction.

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Aragon L, Aragón L, Aragón L, Aragón Let al., 2011, A Double Lock on Sister Chromatids by Cohesin, MOLECULAR CELL, Vol: 44, Pages: 5-6, ISSN: 1097-2765

In this issue of Molecular Cell, Farcas et al. (2011) demonstrate that intertwining between sister chromatids at metaphase is much more significant than previously thought and, remarkably, show that it depends on cohesin.

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Baxter J, Sen N, Lopez Martinez V, Monturus De Carandini ME, Schvartzman JB, Diffley JFX, Aragon L, Baxter J, Sen N, Martínez VL, De Carandini MEM, Schvartzman JB, Diffley JFX, Aragón L, Baxter J, Sen N, Martínez VL, De Carandini MEM, Schvartzman JB, Diffley JFX, Aragón L, Baxter J, Sen N, Martinez VL, De Carandini MEM, Schvartzman JB, Diffley JFX, Aragon Let al., 2011, Positive Supercoiling of Mitotic DNA Drives Decatenation by Topoisomerase II in Eukaryotes, SCIENCE, Vol: 331, Pages: 1328-1332, ISSN: 0036-8075

DNA topoisomerase II completely removes DNA intertwining, or catenation, between sister chromatids before they are segregated during cell division. How this occurs throughout the genome is poorly understood. We demonstrate that in yeast, centromeric plasmids undergo a dramatic change in their topology as the cells pass through mitosis. This change is characterized by positive supercoiling of the DNA and requires mitotic spindles and the condensin factor Smc2. When mitotic positive supercoiling occurs on decatenated DNA, it is rapidly relaxed by topoisomerase II. However, when positive supercoiling takes place in catenated plasmid, topoisomerase II activity is directed toward decatenation of the molecules before relaxation. Thus, a topological change on DNA drives topoisomerase II to decatenate molecules during mitosis, potentially driving the full decatenation of the genome.

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Clemente-Blanco A, Sen N, Mayan-Santos M, Sacristan MP, Graham B, Jarmuz A, Giess A, Webb E, Game L, Eick D, Bueno A, Merkenschlager M, Aragon L, Clemente-Blanco A, Sen N, Mayan-Santos M, Sacristán MP, Graham B, Jarmuz A, Giess A, Webb E, Game L, Eick D, Bueno A, Merkenschlager M, Aragón L, Clemente-Blanco A, Sen N, Mayan-Santos M, Sacristán MP, Graham B, Jarmuz A, Giess A, Webb E, Game L, Eick D, Bueno A, Merkenschlager M, Aragón L, Clemente-Blanco A, Sen N, Mayan-Santos M, Sacristán MP, Graham B, Jarmuz A, Giess A, Webb E, Game L, Eick D, Bueno A, Merkenschlager M, Aragón Let al., 2011, Cdc14 phosphatase promotes segregation of telomeres through repression of RNA polymerase II transcription, NATURE CELL BIOLOGY, Vol: 13, Pages: 1450-U163, ISSN: 1465-7392

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.

JOURNAL ARTICLE

Farmer S, San-Segundo PA, Aragon L, Farmer S, San-Segundo PA, Aragón L, Farmer S, San-Segundo PA, Aragón L, Farmer S, San-Segundo PA, Aragón Let al., 2011, The Smc5-Smc6 Complex Is Required to Remove Chromosome Junctions in Meiosis, PLOS ONE, Vol: 6, Pages: e20948-e20948, ISSN: 1932-6203

Meiosis, a specialized cell division with a single cycle of DNA replication round and two consecutive rounds of nuclear segregation, allows for the exchange of genetic material between parental chromosomes and the formation of haploid gametes. The structural maintenance of chromosome (SMC) proteins aid manipulation of chromosome structures inside cells. Eukaryotic SMC complexes include cohesin, condensin and the Smc5-Smc6 complex. Meiotic roles have been discovered for cohesin and condensin. However, although Smc5-Smc6 is known to be required for successful meiotic divisions, the meiotic functions of the complex are not well understood. Here we show that the Smc5-Smc6 complex localizes to specific chromosome regions during meiotic prophase I. We report that meiotic cells lacking Smc5-Smc6 undergo catastrophic meiotic divisions as a consequence of unresolved linkages between chromosomes. Surprisingly, meiotic segregation defects are not rescued by abrogation of Spo11-induced meiotic recombination, indicating that at least some chromosome linkages in smc5-smc6 mutants originate from other cellular processes. These results demonstrate that, as in mitosis, Smc5-Smc6 is required to ensure proper chromosome segregation during meiosis by preventing aberrant recombination intermediates between homologous chromosomes.

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Seitan VC, Hao B, Tachibana-Konwalski K, Lavagnolli T, Mira-Bontenbal H, Brown KE, Teng G, Carroll T, Terry A, Horan K, Marks H, Adams DJ, Schatz DG, Aragon L, Fisher AG, Krangel MS, Nasmyth K, Merkenschlager M, Seitan VC, Hao B, Tachibana-Konwalski K, Lavagnolli T, Mira-Bontenbal H, Brown KE, Teng G, Carroll T, Terry A, Horan K, Marks H, Adams DJ, Schatz DG, Aragon L, Fisher AG, Krangel MS, Nasmyth K, Merkenschlager M, Seitan VC, Hao B, Tachibana-Konwalski K, Lavagnolli T, Mira-Bontenbal H, Brown KE, Teng G, Carroll T, Terry A, Horan K, Marks H, Adams DJ, Schatz DG, Aragon L, Fisher AG, Krangel MS, Nasmyth K, Merkenschlager M, Seitan VC, Hao B, Tachibana-Konwalski K, Lavagnolli T, Mira-Bontenbal H, Brown KE, Teng G, Carroll T, Terry A, Horan K, Marks H, Adams DJ, Schatz DG, Aragon L, Fisher AG, Krangel MS, Nasmyth K, Merkenschlager Met al., 2011, A role for cohesin in T-cell-receptor rearrangement and thymocyte differentiation, NATURE, Vol: 476, Pages: 467-U126, ISSN: 0028-0836

Cohesin enables post-replicative DNA repair and chromosome segregation by holding sister chromatids together from the time of DNA replication in S phase until mitosis. There is growing evidence that cohesin also forms long-range chromosomal cis-interactions and may regulate gene expression in association with CTCF, mediator or tissue-specific transcription factors. Human cohesinopathies such as Cornelia de Lange syndrome are thought to result from impaired non-canonical cohesin functions, but a clear distinction between the cell-division-related and cell-division-independent functions of cohesion--as exemplified in Drosophila--has not been demonstrated in vertebrate systems. To address this, here we deleted the cohesin locus Rad21 in mouse thymocytes at a time in development when these cells stop cycling and rearrange their T-cell receptor (TCR) α locus (Tcra). Rad21-deficient thymocytes had a normal lifespan and retained the ability to differentiate, albeit with reduced efficiency. Loss of Rad21 led to defective chromatin architecture at the Tcra locus, where cohesion-binding sites flank the TEA promoter and the Eα enhancer, and demarcate Tcra from interspersed Tcrd elements and neighbouring housekeeping genes. Cohesin was required for long-range promoter-enhancer interactions, Tcra transcription, H3K4me3 histone modifications that recruit the recombination machinery and Tcra rearrangement. Provision of pre-rearranged TCR transgenes largely rescued thymocyte differentiation, demonstrating that among thousands of potential target genes across the genome, defective Tcra rearrangement was limiting for the differentiation of cohesin-deficient thymocytes. These findings firmly establish a cell-division-independent role for cohesin in Tcra locus rearrangement and provide a comprehensive account of the mechanisms by which cohesin enables cellular differentiation in a well-characterized mammalian system.

JOURNAL ARTICLE

Ullal P, Vilella-Mitjana F, Jarmuz A, Aragon L, Ullal P, Vilella-Mitjana F, Jarmuz A, Aragón L, Ullal P, Vilella-Mitjana F, Jarmuz A, Aragón L, Ullal P, Vilella-Mitjana F, Jarmuz A, Aragón Let al., 2011, Rtt107 Phosphorylation Promotes Localisation to DNA Double-Stranded Breaks (DSBs) and Recombinational Repair between Sister Chromatids (Retracted article. See vol. 12, art no e0176035, 2017), PLOS ONE, Vol: 6, Pages: e20152-e20152, ISSN: 1932-6203

Efficient repair of DNA double-stranded breaks (DSB) requires a coordinated response at the site of lesion. Nucleolytic resection commits repair towards homologous recombination, which preferentially occurs between sister chromatids. DSB resection promotes recruitment of the Mec1 checkpoint kinase to the break. Rtt107 is a target of Mec1 and serves as a scaffold during repair. Rtt107 plays an important role during rescue of damaged replication forks, however whether Rtt107 contributes to the repair of DSBs is unknown. Here we show that Rtt107 is recruited to DSBs induced by the HO endonuclease. Rtt107 phosphorylation by Mec1 and its interaction with the Smc5-Smc6 complex are both required for Rtt107 loading to breaks, while Rtt107 regulators Slx4 and Rtt101 are not. We demonstrate that Rtt107 has an effect on the efficiency of sister chromatid recombination (SCR) and propose that its recruitment to DSBs, together with the Smc5-Smc6 complex is important for repair through the SCR pathway.

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Aragon L, Aragón L, Aragón L, Aragón Let al., 2010, Ribosomal Genes: Safety in Numbers, CURRENT BIOLOGY, Vol: 20, Pages: R368-R370, ISSN: 0960-9822

The presence of inactive units in tandem arrays of ribosomal genes (rDNA) has been linked to increased transcriptional capacity, but a recent study indicates that inactive units are necessary for sister chromatid cohesion and genetic stability of rDNA.

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Baxter J, Aragón L, Baxter J, Aragón L, Baxter J, Aragon Let al., 2010, Physical linkages between sister chromatids and their removal during yeast chromosome segregation., Cold Spring Harb Symp Quant Biol, Vol: 75, Pages: 389-394, ISSN: 0091-7451

The fidelity of chromosome inheritance is of paramount importance to all living organisms. In eukaryotic cells, the strategy to ensure physical segregation of chromosomes to daughter cells relies on two basic steps ordered in time: an initial linkage, or cohesion, of sister chromatids and its timely and complete dissolution during anaphase. The current view is that these two basic steps are accomplished around the regulation of a protein complex called cohesin that serves as "clamp brackets" distributed at intervals throughout the genome. However, many of the DNA metabolic activities during interphase also produce physical linking of chromatids. For example, during replication, intertwines between sister chromatids are formed. Here, we review our understanding of the processes that generate physical linkages between chromatids and discuss potential mechanisms that are involved in the removal of such obstacles to the complete physical separation of chromatids at anaphase.

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Bermudez-Lopez M, Ceschia A, de Piccoli G, Colomina N, Pasero P, Aragon L, Torres-Rosell J, Bermúdez-López M, Ceschia A, de Piccoli G, Colomina N, Pasero P, Aragón L, Torres-Rosell J, Bermúdez-López M, Ceschia A, de Piccoli G, Colomina N, Pasero P, Aragón L, Torres-Rosell J, Bermudez-Lopez M, Ceschia A, de Piccoli G, Colomina N, Pasero P, Aragon L, Torres-Rosell Jet al., 2010, The Smc5/6 complex is required for dissolution of DNA-mediated sister chromatid linkages, NUCLEIC ACIDS RESEARCH, Vol: 38, Pages: 6502-6512, ISSN: 0305-1048

Mitotic chromosome segregation requires the removal of physical connections between sister chromatids. In addition to cohesin and topological entrapments, sister chromatid separation can be prevented by the presence of chromosome junctions or ongoing DNA replication. We will collectively refer to them as DNA-mediated linkages. Although this type of structures has been documented in different DNA replication and repair mutants, there is no known essential mechanism ensuring their timely removal before mitosis. Here, we show that the dissolution of these connections is an active process that requires the Smc5/6 complex, together with Mms21, its associated SUMO-ligase. Failure to remove DNA-mediated linkages causes gross chromosome missegregation in anaphase. Moreover, we show that Smc5/6 is capable to dissolve them in metaphase-arrested cells, thus restoring chromosome resolution and segregation. We propose that Smc5/6 has an essential role in the removal of DNA-mediated linkages to prevent chromosome missegregation and aneuploidy.

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Mayan M, Aragon L, Mayan M, Aragón L, Mayan M, Aragón L, Mayan M, Aragón Let al., 2010, Cis-interactions between non-coding ribosomal spacers dependent on RNAP-II separate RNAP-I and RNAP-III transcription domains, CELL CYCLE, Vol: 9, Pages: 4328-4337, ISSN: 1538-4101

Ribosome biogenesis requires transcription of structural RNAs. In budding yeast, ribosomal units contain both 35S and 5S RNA genes separated by intergenic spacer sequences (IGS) that are transcribed by RNAP-II. IGS transcripts cause instability by promoting unequal sister chromatid recombination between repeats and are thus rapidly degraded by the exosome. Whether RNAP-II within IGS regions plays any functional role is unknown. Here we demonstrate that the bulk of RNAP-II bound to IGS sites is blocked for elongation and hence remains in a poised or stalled configuration. We describe a novel role for these stalled RNAP-II complexes in the formation of cis-interactions between the IGS of rDNA. We show that this function separates 35S and 5S RNA genes into polymerase-specific chromatin loops and demonstrate that removal of stalled RNAP-II complexes causes displacement of RNAP-III from the 5S gene region and transcriptional downregulation of 5S rRNA by spreading of RNAP-I. We conclude that stalled RNAP-II plays an active role in the cis-organisation of ribosomal repeats providing domains of polymerase specificity in the nucleolar transcription environment.

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Clemente-Blanco A, Mayan-Santos M, Schneider DA, Machin F, Jarmuz A, Tschochner H, Aragon L, Clemente-Blanco A, Mayán-Santos M, Schneider DA, Machín F, Jarmuz A, Tschochner H, Aragón L, Clemente-Blanco A, Mayán-Santos M, Schneider DA, Machín F, Jarmuz A, Tschochner H, Aragón L, Clemente-Blanco A, Mayán-Santos M, Schneider DA, Machín F, Jarmuz A, Tschochner H, Aragón Let al., 2009, Cdc14 inhibits transcription by RNA polymerase I during anaphase, NATURE, Vol: 458, Pages: 219-U8, ISSN: 0028-0836

Chromosome condensation and the global repression of gene transcription are features of mitosis in most eukaryotes. The logic behind this phenomenon is that chromosome condensation prevents the activity of RNA polymerases. In budding yeast, however, transcription was proposed to be continuous during mitosis. Here we show that Cdc14, a protein phosphatase required for nucleolar segregation and mitotic exit, inhibits transcription of yeast ribosomal genes (rDNA) during anaphase. The phosphatase activity of Cdc14 is required for RNA polymerase I (Pol I) inhibition in vitro and in vivo. Moreover Cdc14-dependent inhibition involves nucleolar exclusion of Pol I subunits. We demonstrate that transcription inhibition is necessary for complete chromosome disjunction, because ribosomal RNA (rRNA) transcripts block condensin binding to rDNA, and show that bypassing the role of Cdc14 in nucleolar segregation requires in vivo degradation of nascent transcripts. Our results show that transcription interferes with chromosome condensation, not the reverse. We conclude that budding yeast, like most eukaryotes, inhibit Pol I transcription before segregation as a prerequisite for chromosome condensation and faithful genome separation.

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Conde F, Refolio E, Cordon-Preciado V, Cortes-Ledesma F, Aragon L, Aguilera A, San-Segundo PA, Conde F, Refolio E, Cordón-Preciado V, Cortés-Ledesma F, Aragón L, Aguilera A, San-Segundo PA, Conde F, Refolio E, Cordón-Preciado V, Cortés-Ledesma F, Aragón L, Aguilera A, San-Segundo PA, Conde F, Refolio E, Cordon-Preciado V, Cortes-Ledesma F, Aragon L, Aguilera A, San-Segundo PAet al., 2009, The Dot1 Histone Methyltransferase and the Rad9 Checkpoint Adaptor Contribute to Cohesin-Dependent Double-Strand Break Repair by Sister Chromatid Recombination in Saccharomyces cerevisiae, GENETICS, Vol: 182, Pages: 437-446, ISSN: 0016-6731

Genomic integrity is threatened by multiple sources of DNA damage. DNA double-strand breaks (DSBs) are among the most dangerous types of DNA lesions and can be generated by endogenous or exogenous agents, but they can arise also during DNA replication. Sister chromatid recombination (SCR) is a key mechanism for the repair of DSBs generated during replication and it is fundamental for maintaining genomic stability. Proper repair relies on several factors, among which histone modifications play important roles in the response to DSBs. Here, we study the role of the histone H3K79 methyltransferase Dot1 in the repair by SCR of replication-dependent HO-induced DSBs, as a way to assess its function in homologous recombination. We show that Dot1, the Rad9 DNA damage checkpoint adaptor, and phosphorylation of histone H2A (gammaH2A) are required for efficient SCR. Moreover, we show that Dot1 and Rad9 promote DSB-induced loading of cohesin onto chromatin. We propose that recruitment of Rad9 to DSB sites mediated by gammaH2A and H3K79 methylation contributes to DSB repair via SCR by regulating cohesin binding to damage sites. Therefore, our results contribute to an understanding of how different chromatin modifications impinge on DNA repair mechanisms, which are fundamental for maintaining genomic stability.

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De Piccoli G, Torres-Rosell J, Aragon L, De Piccoli G, Torres-Rosell J, Aragón L, De Piccoli G, Torres-Rosell J, Aragón L, Piccoli GD, Torres-Rosell J, Aragón Let al., 2009, The unnamed complex: what do we know about Smc5-Smc6?, CHROMOSOME RESEARCH, Vol: 17, Pages: 251-263, ISSN: 0967-3849

The structural maintenance of chromosome (SMC) proteins constitute the cores of three protein complexes involved in chromosome metabolism; cohesin, condensin and the Smc5-Smc6 complex. While the roles of cohesin and condensin in sister chromatid cohesion and chromosome condensation respectively have been described, the cellular function of Smc5-Smc6 is as yet not understood, consequently the less descriptive name. The complex is involved in a variety of DNA repair pathways. It contains activities reminiscent of those described for cohesin and condensin, as well as several DNA helicases and endonucleases. It is required for sister chromatid recombination, and smc5-smc6 mutants suffer from the accumulation of unscheduled recombination intermediates. The complex contains a SUMO-ligase and potentially an ubiquitin-ligase; thus Smc5-Smc6 might presently have a dull name, but it seems destined to be recognized as a key player in the maintenance of chromosome stability. In this review we summarize our present understanding of this enigmatic protein complex.

JOURNAL ARTICLE

Dulev S, Aragon L, Strunnikov A, Dulev S, Aragon L, Strunnikov A, Dulev S, Aragon L, Strunnikov A, Dulev Set al., 2008, Unreplicated DNA in mitosis precludes condensin binding and chromosome condensation in S. cerevisiae, FRONTIERS IN BIOSCIENCE, Vol: 13, Pages: 5838-5846, ISSN: 1093-9946

Condensin is the core activity responsible for chromosome condensation in mitosis. In the yeast S. cerevisiae, condensin binding is enriched at the regions where DNA replication terminates. Therefore, we investigated whether DNA replication completion determines the condensin-binding proficiency of chromatin. In order to fulfill putative mitotic requirements for condensin activity we analyzed chromosome condensation and condensin binding to unreplicated chromosomes in mitosis. For this purpose we used pGAL:CDC6 cdc15-ts cells that are known to enter mitosis without DNA replication if CDC6 transcription is repressed prior to S-phase. Both the condensation of nucleolar chromatin and proper condensin targeting to rDNA sites failed when unreplicated chromosomes were driven in mitosis. We propose that the DNA replication results in structural and/or biochemical changes to replicated chromatin, which are required for two-phase condensin binding and proper chromosome condensation.

JOURNAL ARTICLE

Hwang J-Y, Smith S, Ceschia A, Torres-Rosell J, Aragon L, Myung K, Hwang J-Y, Smith S, Ceschia A, Torres-Rosell J, Aragon L, Myung K, Hwang J-Y, Smith S, Ceschia A, Torres-Rosell J, Aragon L, Myung K, Hwang J-Y, Smith S, Ceschia A, Torres-Rosell J, Aragon L, Myung Ket al., 2008, Smc5-Smc6 complex suppresses gross chromosomal rearrangements mediated by break-induced replications, DNA REPAIR, Vol: 7, Pages: 1426-1436, ISSN: 1568-7864

Translocations in chromosomes alter genetic information. Although the frequent translocations observed in many tumors suggest the altered genetic information by translocation could promote tumorigenesis, the mechanisms for how translocations are suppressed and produced are poorly understood. The smc6-9 mutation increased the translocation class gross chromosomal rearrangement (GCR). Translocations produced in the smc6-9 strain are unique because they are non-reciprocal and dependent on break-induced replication (BIR) and independent of non-homologous end joining. The high incidence of translocations near repetitive sequences such as delta sequences, ARS, tRNA genes, and telomeres in the smc6-9 strain indicates that Smc5-Smc6 suppresses translocations by reducing DNA damage at repetitive sequences. Synergistic enhancements of translocations in strains defective in DNA damage checkpoints by the smc6-9 mutation without affecting de novo telomere addition class GCR suggest that Smc5-Smc6 defines a new pathway to suppress GCR formation.

JOURNAL ARTICLE

Hwang J-Y, Smith S, Torres-Rosell J, Aragon L, Myung K, Hwang JY, Smith S, Torres-Rosell J, Aragon L, Myung Ket al., 2008, Smc5-Smc6 regulate gross chromosomal rearrangements and mediate DNA double-strand-break repair, Joint Conference of the 33rd FEBS Congress/11th IUBMB Conference, Publisher: BLACKWELL PUBLISHING, Pages: 113-113, ISSN: 1742-464X

CONFERENCE PAPER

Parelho V, Hadjur S, Spivakov M, Leleu M, Sauer S, Gregson HC, Jarmuz A, Canzonetta C, Webster Z, Nesterova T, Cobb BS, Yokomori K, Dillon N, Aragon L, Fisher AG, Merkenschiager M, Parelho V, Hadjur S, Spivakov M, Leleu M, Sauer S, Gregson HC, Jarmuz A, Canzonetta C, Webster Z, Nesterova T, Cobb BS, Yokomori K, Dillon N, Aragon L, Fisher AG, Merkenschlager M, Parelho V, Hadjur S, Spivakov M, Leleu M, Sauer S, Gregson HC, Jarmuz A, Canzonetta C, Webster Z, Nesterova T, Cobb BS, Yokomori K, Dillon N, Aragon L, Fisher AG, Merkenschlager M, Parelho V, Hadjur S, Spivakov M, Leleu M, Sauer S, Gregson HC, Jarmuz A, Canzonetta C, Webster Z, Nesterova T, Cobb BS, Yokomori K, Dillon N, Aragon L, Fisher AG, Merkenschlager Met al., 2008, Cohesins functionally associate with CTCF on mammalian chromosome arms, CELL, Vol: 132, Pages: 422-433, ISSN: 0092-8674

Cohesins mediate sister chromatid cohesion, which is essential for chromosome segregation and postreplicative DNA repair. In addition, cohesins appear to regulate gene expression and enhancer-promoter interactions. These noncanonical functions remained unexplained because knowledge of cohesin-binding sites and functional interactors in metazoans was lacking. We show that the distribution of cohesins on mammalian chromosome arms is not driven by transcriptional activity, in contrast to S. cerevisiae. Instead, mammalian cohesins occupy a subset of DNase I hypersensitive sites, many of which contain sequence motifs resembling the consensus for CTCF, a DNA-binding protein with enhancer blocking function and boundary-element activity. We find cohesins at most CTCF sites and show that CTCF is required for cohesin localization to these sites. Recruitment by CTCF suggests a rationale for noncanonical cohesin functions and, because CTCF binding is sensitive to DNA methylation, allows cohesin positioning to integrate DNA sequence and epigenetic state.

JOURNAL ARTICLE

Torres-Rosell J, De Piccoli G, Aragon L, Torres-Rosell J, De Piccoli G, Aragón L, Torres-Rosell J, De Piccoli G, Aragón L, Torres-Rosell J, De Piccoli G, Aragón Let al., 2007, Can eukaryotic cells monitor the presence of unreplicated DNA?, CELL DIVISION, Vol: 2, Pages: 19-19, ISSN: 1747-1028

Completion of DNA replication before mitosis is essential for genome stability and cell viability. Cellular controls called checkpoints act as surveillance mechanisms capable of detecting errors and blocking cell cycle progression to allow time for those errors to be corrected. An important question in the cell cycle field is whether eukaryotic cells possess mechanisms that monitor ongoing DNA replication and make sure that all chromosomes are fully replicated before entering mitosis, that is whether a replication-completion checkpoint exists. From recent studies with smc5-smc6 mutants it appears that yeast cells can enter anaphase without noticing that replication in the ribosomal DNA array was unfinished. smc5-smc6 mutants are proficient in all known cellular checkpoints, namely the S phase checkpoint, DNA-damage checkpoint, and spindle checkpoint, thus suggesting that none of these checkpoints can monitor the presence of unreplicated segments or the unhindered progression of forks in rDNA. Therefore, these results strongly suggest that normal yeast cells do not contain a DNA replication-completion checkpoint.

JOURNAL ARTICLE

Torres-Rosell J, De Piccoli G, Cordon-Preciado V, Farmer S, Jamuz A, Machin F, Pasero P, Lisby M, Haber JE, Aragon L, Torres-Rosell J, De Piccoli G, Cordon-Preciado V, Farmer S, Jarmuz A, Machin F, Pasero P, Lisby M, Haber JE, Aragón L, Torres-Rosell J, De Piccoli G, Cordon-Preciado V, Farmer S, Jarmuz A, Machin F, Pasero P, Lisby M, Haber JE, Aragón L, Torres-Rosell J, De Piccoli G, Cordon-Preciado V, Farmer S, Jarmuz A, Machin F, Pasero P, Lisby M, Haber JE, Aragon Let al., 2007, Anaphase onset before complete DNA replication with intact checkpoint responses, SCIENCE, Vol: 315, Pages: 1411-1415, ISSN: 0036-8075

Cellular checkpoints prevent mitosis in the presence of stalled replication forks. Whether checkpoints also ensure the completion of DNA replication before mitosis is unknown. Here, we show that in yeast smc5-smc6 mutants, which are related to cohesin and condensin, replication is delayed, most significantly at natural replication-impeding loci like the ribosomal DNA gene cluster. In the absence of Smc5-Smc6, chromosome nondisjunction occurs as a consequence of mitotic entry with unfinished replication despite intact checkpoint responses. Eliminating processes that obstruct replication fork progression restores the temporal uncoupling between replication and segregation in smc5-smc6 mutants. We propose that the completion of replication is not under the surveillance of known checkpoints.

JOURNAL ARTICLE

Torres-Rosell J, Sunjevaric I, De Piccoli G, Sacher M, Eckert-Boulet N, Reid R, Jentsch S, Rothstein R, Aragon L, Lisby M, Torres-Rosell J, Sunjevaric I, De Piccoli G, Sacher M, Eckert-Boulet N, Reid R, Jentsch S, Rothstein R, Aragón L, Lisby M, Torres-Rosell J, Sunjevaric I, De Piccoli G, Sacher M, Eckert-Boulet N, Reid R, Jentsch S, Rothstein R, Aragón L, Lisby M, Torres-Rosell J, Sunjevaric I, De Piccoli G, Sacher M, Eckert-Boulet N, Reid R, Jentsch S, Rothstein R, Aragón L, Lisby Met al., 2007, The Smc5-Smc6 complex and SUMO modification of Rad52 regulates recombinational repair at the ribosomal gene locus, NATURE CELL BIOLOGY, Vol: 9, Pages: 923-U72, ISSN: 1465-7392

Homologous recombination (HR) is crucial for maintaining genome integrity by repairing DNA double-strand breaks (DSBs) and rescuing collapsed replication forks. In contrast, uncontrolled HR can lead to chromosome translocations, loss of heterozygosity, and deletion of repetitive sequences. Controlled HR is particularly important for the preservation of repetitive sequences of the ribosomal gene (rDNA) cluster. Here we show that recombinational repair of a DSB in rDNA in Saccharomyces cerevisiae involves the transient relocalization of the lesion to associate with the recombination machinery at an extranucleolar site. The nucleolar exclusion of Rad52 recombination foci entails Mre11 and Smc5-Smc6 complexes and depends on Rad52 SUMO (small ubiquitin-related modifier) modification. Remarkably, mutations that abrogate these activities result in the formation of Rad52 foci within the nucleolus and cause rDNA hyperrecombination and the excision of extrachromosomal rDNA circles. Our study also suggests a key role of sumoylation for nucleolar dynamics, perhaps in the compartmentalization of nuclear activities.

JOURNAL ARTICLE

Clemente-Blanco A, Gonzalez-Novo A, Machin F, Caballero-Lima D, Aragon L, Sanchez M, de Aldana CRV, Jimenez J, Correa-Bordes J, Clemente-Blanco A, González-Novo A, Machín F, Caballero-Lima D, Aragón L, Sánchez M, de Aldana CRV, Jiménez J, Correa-Bordes J, Clemente-Blanco A, González-Novo A, Machín F, Caballero-Lima D, Aragón L, Sánchez M, de Aldana CRV, Jiménez J, Correa-Bordes J, Clemente-Blanco A, Clemente-Blanco A, Gonzalez-Novo A, Machin F, Caballero-Lima D, Aragon L, Sanchez M, Vazquez de Aldana C, Jimenez J, Correa-Bordes Jet al., 2006, The Cdc14p phosphatase affects late cell-cycle events and morphogenesis in Candida albicans, JOURNAL OF CELL SCIENCE, Vol: 119, Pages: 1130-1143, ISSN: 0021-9533

We have characterized the CDC14 gene, which encodes a dual-specificity protein phosphatase in Candida albicans, and demonstrated that its deletion results in defects in cell separation, mitotic exit and morphogenesis. The C. albicans cdc14delta mutants formed large aggregates of cells that resembled those found in ace2-null strains. In cdc14delta cells, expression of Ace2p target genes was reduced and Ace2p did not accumulate specifically in daughter nuclei. Taken together, these results imply that Cdc14p is required for the activation and daughter-specific nuclear accumulation of Ace2p. Consistent with a role in cell separation, Cdc14p was targeted to the septum region during the M-G1 transition in yeast-form cells. Interestingly, hypha-inducing signals abolished the translocation of Cdc14p to the division plate, and this regulation depended on the cyclin Hgc1p, since hgc1delta mutants were able to accumulate Cdc14p in the septum region of the germ tubes. In addition to its role in cytokinesis, Cdc14p regulated mitotic exit, since synchronous cultures of cdc14delta cells exhibited a severe delay in the destruction of the mitotic cyclin Clb2p. Finally, deletion of CDC14 resulted in decreased invasion of solid agar medium and impaired true hyphal growth.

JOURNAL ARTICLE

De Piccoli G, Cortes-Ledesma F, Ira G, Torres-Rosell J, Uhle S, Farmer S, Hwang J-Y, Machin F, Ceschia A, McAleenan A, Cordon-Preciado V, Clemente-Blanco A, Vilella-Mitjana F, Ullal P, Jarmuz A, Leitao B, Bressan D, Dotiwala F, Papusha A, Zhao X, Myung K, Haber JE, Aguilera A, Aragon L, De Piccoli G, Cortes-Ledesma F, Ira G, Torres-Rosell J, Uhle S, Farmer S, Hwang J-Y, Machin F, Ceschia A, McAleenan A, Cordon-Preciado V, Clemente-Blanco A, Vilella-Mitjana F, Ullal P, Jarmuz A, Leitao B, Bressan D, Dotiwala F, Papusha A, Zhao X, Myung K, Haber JE, Aguilera A, Aragón L, De Piccoli G, Cortes-Ledesma F, Ira G, Torres-Rosell J, Uhle S, Farmer S, Hwang J-Y, Machin F, Ceschia A, McAleenan A, Cordon-Preciado V, Clemente-Blanco A, Vilella-Mitjana F, Ullal P, Jarmuz A, Leitao B, Bressan D, Dotiwala F, Papusha A, Zhao X, Myung K, Haber JE, Aguilera A, Aragón L, De Piccoli G, Cortes-Ledesma F, Ira G, Torres-Rosell J, Uhle S, Farmer S, Hwang J-Y, Machin F, Ceschia A, McAleenan A, Cordon-Preciado V, Clemente-Blanco A, Vilella-Mitjana F, Ullal P, Jarmuz A, Leitao B, Bressan D, Dotiwala F, Papusha A, Zhao X, Myung K, Haber JE, Aguilera A, Aragón Let al., 2006, Smc5-Smc6 mediate DNA double-strand-break repair by promoting sister-chromatid recombination, NATURE CELL BIOLOGY, Vol: 8, Pages: 1032-U118, ISSN: 1465-7392

DNA double-strand breaks (DSB) can arise during DNA replication, or after exposure to DNA-damaging agents, and their correct repair is fundamental for cell survival and genomic stability. Here, we show that the Smc5-Smc6 complex is recruited to DSBs de novo to support their repair by homologous recombination between sister chromatids. In addition, we demonstrate that Smc5-Smc6 is necessary to suppress gross chromosomal rearrangements. Our findings show that the Smc5-Smc6 complex is essential for genome stability as it promotes repair of DSBs by error-free sister-chromatid recombination (SCR), thereby suppressing inappropriate non-sister recombination events.

JOURNAL ARTICLE

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