47 results found
Bermudez-Lopez M, Aragon L, 2017, Smc5/6 complex regulates Sgs1 recombination functions, CURRENT GENETICS, Vol: 63, Pages: 381-388, ISSN: 0172-8083
Ramos F, Leonard J, Clemente-Blanco A, et al., 2017, Cdc14 and Chromosome Condensation: Evaluation of the Recruitment of Condensin to Genomic Regions., Methods Mol Biol, Vol: 1505, Pages: 229-243
Chromosome condensation is an essential morphological event required for successful DNA segregation during mitosis. The high level of genome compaction achieved during this process is attained by the evolutionary conserved condensin complex. Recently, several lines of evidences have demonstrated that the mitotic phosphatase Cdc14 is required to ensure condensin loading onto chromosomes. To date several approaches have been used in order to characterize condensin activity and regulation, however these techniques are time-consuming and require complex equipment. In this chapter we described an easy and reliable protocol to analyze Cdc14-dependent condensin loading onto specific genomic DNA regions by using a chromatin immunoprecipitation (ChIP) technique.
Bermúdez-López M, Aragón L, 2016, Detection of Cohesin SUMOylation In Vivo., Methods Mol Biol, Vol: 1515, Pages: 55-64
Cohesin is a protein complex with key roles in chromosome biology, from chromatid segregation to DNA repair. Cohesin function is regulated by several posttranslational modifications, including phosphorylation, acetylation, ubiquitylation, and SUMOylation. Recent studies have shown that cohesin SUMOylation is essential for sister chromatid cohesion during normal cell cycle and in response to DNA damage. Posttranslational modification by the small ubiquitin-like modifier (SUMO) is a field in expansion, however, detecting SUMOylation can be challenging because the amount of modified substrates are usually low and de-conjugation during sample preparation often occurs. In this chapter we describe a method that can be adapted to different model organisms, and substrates to detect SUMOylation. We focus on cohesin and show that SUMOylation indeed occurs in most of the subunits of budding yeast cohesin.
Sen N, Leonard J, Torres R, et al., 2016, Physical proximity of sister chromatids promotestop2-dependent intertwining, Molecular Cell, Vol: 64, Pages: 134-147, ISSN: 1097-4164
Sister chromatid intertwines (SCIs), or catenanes, aretopological links between replicated chromatids thatinterfere with chromosome segregation. The forma-tion of SCIs is thought to be a consequence of forkswiveling during DNA replication, and their removalis thought to occur because of the intrinsic featureof type II topoisomerases (Top2) to simplify DNAtopology. Here, we report that SCIs are also formedindependently of DNA replication during G2/M byTop2-dependent concatenation of cohesed chroma-tids due to their physical proximity. We demonstratethat, in contrast to G2/M, Top2 removes SCIs from co-hesedchromatidsattheanaphaseonset.Importantly,SCI removal in anaphase requires condensin and co-incides with the hyperactivation of condensin DNAsupercoiling activity. This is consistent with the long-standing proposal that condensin provides a bias inTop2functiontowarddecatenation.Acomprehensivemodel for the formation and resolution of toxic SCIentanglements on eukaryotic genomes is proposed.
Bermudez-Lopez M, Villoria MT, Esteras M, et al., 2016, Sgs1’s roles in DNA end resection, HJdissolution, and crossover suppressionrequire a two-step SUMO regulationdependent on Smc5/6, Genes & Development, Vol: 30, Pages: 1339-1356, ISSN: 1549-5477
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.
Leonard J, Sen N, Torres R, et al., 2015, Condensin Relocalization from Centromeres to Chromosome Arms Promotes Top2 Recruitment during Anaphase, Cell Reports, Vol: 13, Pages: 2336-2344, ISSN: 2211-1247
Patel A, Aragon L, 2015, A Double Strand Break during Telophase Is Repaired with Homologous Recombination Despite the Absence of an Available Sister Chromatid, Blood, Vol: 126, Pages: 2421-2421, ISSN: 0006-4971
Background:Chromosomal breakage results from a DNA double strand break (DSB), and is repaired to maintain and restore genetic integrity, principally through two major pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ). HR is initiated by nucleolytic resection of a DSB in the presence of cyclin-dependent kinase 1 (Cdk1) activity. DSB repair through HR is dependent on Rad52, and can be error-free when a sister chromatid is used as a template for repair. However, HR is mutagenic when any other template is used for repair. Loss of nucleotides adjacent to the DSB is a feature of repair through NHEJ. There is co-relation between Cdk1 activity and the presence of a sister chromatid. The research question was, in addition to Cdk1 activity is the presence of an intact sister chromatid a requirement to initiate DSB repair with the HR pathway.Methods:Cdk1 activity peaks during mitosis in the presence of an intact sister chromatid. To study DSB resection and repair in cells arrested in either mitotic metaphase or telophase when Cdk1-Clb2 was active, conditional alleles were constructed in a eukaryotic haploid budding yeast model of HR. The model permitted simultaneous induction of a single site-specific DSB in cells that were synchronised to a phase of the cell division cycle. Physical monitoring of the kinetics of DSB formation, nucleolytic resection of adjacent DNA, and DSB repair, was achieved by probing Southern membranes after restriction enzyme digestion of extracted genomic DNA from time courses.Results:Sister chromatids were segregated during telophase arrest induced by either Cdc14 or Cdc15 depletion. Metaphase arrest was achieved with Cdc20 depletion, either directly, or indirectly by activation of the spindle assembly checkpoint by inhibition of microtubule polymerisation. Sister chromatids were unsegregated and physically attached through cohesin during metaphase.The absence of an intact sister chromatid did not prevent DSB repair with the
Mayán MD, Aragón L, 2014, Chromosome conformation capture (3C) of tandem arrays in yeast., Methods Mol Biol, Vol: 1205, Pages: 219-229
Studying interphase chromosome arrangements at the molecular level can provide important details on the function and coordination of many metabolic processes that take place on DNA, such as transcription or DNA repair. The chromosome conformation capture (3C) methodology was originally developed in yeast to study the interphase organization of a single-yeast chromosome. 3C assays allow the identification of physical interactions between distant DNA segments and thus provide detailed information on the folding of chromatin in the native cellular state. Since its initial development, the technique has been used to advance our understanding of the folding of gene loci and has yielded important discoveries, like the demonstration that distant transcriptional regulatory elements interact with their target promoters through chromatin loops. The 3C method uses formaldehyde cross-linking to covalently link interacting chromatin segments in intact cells. After chromatin digestion and ligation of cross-linked fragments, the frequency of interaction between distant DNA elements can be quantified. However, the design, analysis, and controls used are critical when using 3C. In this chapter, we describe the general protocol for 3C analysis and demonstrate it through analysis of interactions in repetitive sequences of the ribosomal gene array of Saccharomyces cerevisiae.
Patel A, 2014, The role of the sister chromatid during repair of a DNA double-strand break
Chromosomal breaks are extremely cytotoxic and can occur during normal cell metabolism, and after exposure to exogenous DNA damaging agents. Double strand breaks (DSBs) are repaired to maintain and restore genetic integrity, principally through two major pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ). HR can be error-free when sister chromatids are used as a template for repair, and is initiated by nucleolytic resection of the DSB. Cyclin-dependent kinase 1 (Cdk1) activity is crucial to promote HR. As Cdk1 activity and the sister chromatid are only present during certain cell division cycle stages, this study investigated whether in addition to Cdk1 activity the presence of an intact sister chromatid is a requirement to initiate HR. Conditional alleles that arrest the cell division cycle with separated sister chromatids and high Cdk1 activity were constructed in budding yeast and used to investigate this possibility. This study has found that HR occurs with segregated sister chromatids during telophase, at a time when mitotic Cdk1 activity is high. HR is also less efficient during metaphase if microtubule function is impaired. Overall, the availability of the sister chromatid is not an additional requirement to mitotic Cdk1 activity to promote DSB repair with the HR pathway.
Garcia-Luis J, Clemente-Blanco A, Aragon L, et al., 2014, Cdc14 targets the Holliday junction resolvase Yen1 to the nucleus in early anaphase, CELL CYCLE, Vol: 13, Pages: 1392-1399, ISSN: 1538-4101
Aragon L, Martinez-Perez E, Merkenschlager M, 2013, Condensin, cohesin and the control of chromatin states, CURRENT OPINION IN GENETICS & DEVELOPMENT, Vol: 23, Pages: 204-211, ISSN: 0959-437X
McAleenan A, Clemente-Blanco A, Cordon-Preciado V, et al., 2013, Post-replicative repair involves separase-dependent removal of the kleisin subunit of cohesin, NATURE, Vol: 493, Pages: 250-U270, ISSN: 0028-0836
McAleenan A, Cordon-Preciado V, Clemente-Blanco A, et 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
Baxter J, Aragon L, 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
Quevedo O, Garcia-Luis J, Matos-Perdomo E, et al., 2012, Nondisjunction of a Single Chromosome Leads to Breakage and Activation of DNA Damage Checkpoint in G2, PLOS GENETICS, Vol: 8, ISSN: 1553-7404
Clemente-Blanco A, Sen N, Mayan-Santos M, et 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 exit2,3 and for segregation of repetitive regions4. 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 mutants4 correlate with the presence of subtelomeric Y′ elements and can be rescued by transcriptional inhibition of RNA polymerase II.
Aragon L, 2011, A Double Lock on Sister Chromatids by Cohesin, MOLECULAR CELL, Vol: 44, Pages: 5-6, ISSN: 1097-2765
Seitan VC, Hao B, Tachibana-Konwalski K, et 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 mitosis1. There is growing evidence that cohesin also forms long-range chromosomal cis-interactions2,3,4 and may regulate gene expression2,3,4,5,6,7,8,9,10 in association with CTCF8,9, mediator4 or tissue-specific transcription factors10. Human cohesinopathies such as Cornelia de Lange syndrome are thought to result from impaired non-canonical cohesin functions7, but a clear distinction between the cell-division-related and cell-division-independent functions of cohesion—as exemplified in Drosophila11,12,13—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 machinery14,15 and Tcra rearrangement. Provision of pre-rearranged TCR transgenes largely rescued thymocyte differentiation, demonstrating that among thousands of potential target genes across the genome4,8,9,10, 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 mammali
Farmer S, San-Segundo PA, Aragon L, 2011, The Smc5-Smc6 Complex Is Required to Remove Chromosome Junctions in Meiosis, PLOS ONE, Vol: 6, ISSN: 1932-6203
Ullal P, Vilella-Mitjana F, Jarmuz A, et 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, ISSN: 1932-6203
Baxter J, Sen N, Lopez Martinez V, et al., 2011, Positive Supercoiling of Mitotic DNA Drives Decatenation by Topoisomerase II in Eukaryotes, SCIENCE, Vol: 331, Pages: 1328-1332, ISSN: 0036-8075
Mayan M, Aragon L, 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
Bermudez-Lopez M, Ceschia A, de Piccoli G, et 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
Aragon L, 2010, Ribosomal Genes: Safety in Numbers, CURRENT BIOLOGY, Vol: 20, Pages: R368-R370, ISSN: 0960-9822
Baxter J, Aragón L, 2010, Physical linkages between sister chromatids and their removal during yeast chromosome segregation., Cold Spring Harb Symp Quant Biol, Vol: 75, Pages: 389-394
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.
Conde F, Refolio E, Cordon-Preciado V, et 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
Clemente-Blanco A, Mayan-Santos M, Schneider DA, et al., 2009, Cdc14 inhibits transcription by RNA polymerase I during anaphase, NATURE, Vol: 458, Pages: 219-U8, ISSN: 0028-0836
De Piccoli G, Torres-Rosell J, Aragon L, 2009, The unnamed complex: what do we know about Smc5-Smc6?, CHROMOSOME RESEARCH, Vol: 17, Pages: 251-263, ISSN: 0967-3849
Hwang J-Y, Smith S, Ceschia A, et al., 2008, Smc5-Smc6 complex suppresses gross chromosomal rearrangements mediated by break-induced replications, DNA REPAIR, Vol: 7, Pages: 1426-1436, ISSN: 1568-7864
Hwang J-Y, Smith S, Torres-Rosell J, et 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
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