Domain within the helicase subunit Mcm4 integrates multiple kinase signals to control DNA replication initiation and fork progression

Eukaryotic DNA synthesis initiates from multiple replication origins and progresses through bidirectional replication forks to ensure efficient duplication of the genome. Temporal control of initiation from origins and regulation of replication fork functions are important aspects for maintaining genome stability. Multiple kinase-signaling pathways are involved in these processes. The Dbf4-dependent Cdc7 kinase (DDK), cyclin-dependent kinase (CDK), and Mec1, the yeast Ataxia telangiectasia mutated/Ataxia telangiectasia mutated Rad3-related checkpoint regulator, all target the structurally disordered N-terminal serine/threonine-rich domain (NSD) of mini-chromosome maintenance subunit 4 (Mcm4), a subunit of the mini-chromosome maintenance (MCM) replicative helicase complex. Using whole-genome replication profile analysis and single-molecule DNA fiber analysis, we show that under replication stress the temporal pattern of origin activation and DNA replication fork progression are altered in cells with mutations within two separate segments of the Mcm4 NSD. The proximal segment of the NSD residing next to the DDK-docking domain mediates repression of late-origin firing by checkpoint signals because in its absence late origins become active despite an elevated DNA damage-checkpoint response. In contrast, the distal segment of the NSD at the N terminus plays no role in the temporal pattern of origin firing but has a strong influence on replication fork progression and on checkpoint signaling. Both fork progression and checkpoint response are regulated by the phosphorylation of the canonical CDK sites at the distal NSD. Together, our data suggest that the eukaryotic MCM helicase contains an intrinsic regulatory domain that integrates multiple signals to coordinate origin activation and replication fork progression under stress conditions

Principles of meiotic chromosome assembly revealed in S. cerevisiae

During meiotic prophase, chromosomes organise into a series of chromatin loops emanating from a proteinaceous axis, but the mechanisms of assembly remain unclear. Here we use Saccharomyces cerevisiae to explore how this elaborate three-dimensional chromosome organisation is linked to genomic sequence. As cells enter meiosis, we observe that strong cohesin-dependent grid-like Hi-C interaction patterns emerge, reminiscent of mammalian interphase organisation, but with distinct regulation. Meiotic patterns agree with simulations of loop extrusion with growth limited by barriers, in which a heterogeneous population of expanding loops develop along the chromosome. Importantly, CTCF, the factor that imposes similar features in mammalian interphase, is absent in S. cerevisiae, suggesting alternative mechanisms of barrier formation. While grid-like interactions emerge independently of meiotic chromosome synapsis, synapsis itself generates additional compaction that matures differentially according to telomere proximity and chromosome size. Collectively, our results elucidate fundamental principles of chromosome assembly and demonstrate the essential role of cohesin within this evolutionarily conserved process

Pluralidad y continuidad: espacios sonoros múltiples en la armonía y la estructura de una composición musical. Alrededor de la obra fragments pour un labyrinthe de cendres.

Tesis realizada en la Maestría Interinstitucional en Música ( Minter ) desarrollada por convenio entre la Universidad de la República (Udelar) y la Universidad Federal de Río Grande del Sur (UFRGS)El presente trabajo se propone desarrollar una reflexión sobre la organización microinterválica de la obra fragments pour un labyrinthe de cendres y sus derivadas, compuestas a partir de la idea de la confrontación y el encuentro entre varios espacios sonoros. El proceso incluye la evaluación del concepto de espacio sonoro, su aplicación en las obras y las posibilidades de una pluralidad de espacios sonoros simultáneos y sucesivos. La generación de una poliespacialidad sonora plantea la problemática notacional así como su realización concreta. Esta poliespacialidad sonora se manifiesta a través del uso melódico: sus consecuencias se analizan en el ámbito tímbrico de los instrumentos reales y virtuales usados en la obra así como en el desarrollo de una armonía integral estática polifacética.Introducción. -- I. El espacio sonoro y su materialización en la composición. -- 1. Una obra de múltiples capas. -- 2. Los espacios sonoros de fragments…, pluralidad e interacción. -- 2.1. Espacio en semitonos. -- 2.2. Espacio en cuartos de tono. -- 2.3. Espacios en séptimos de tono. -- 2.4. Espacio equipentatónico. -- 2.5. Espacio equiheptatónico. -- 2.6. Espacios del gamelan. -- 2.7. Espacio indeterminado. -- 2.8. Superposición de espacios. -- 3. El espacio sonoro, más allá de la escala. -- 3.1. El concepto espacial. -- 3.2. Continuidad o discontinuidad. -- 3.3. Relatividad. -- 4. Los espacios sonoros resultantes en lo concreto de la obra. -- 4.1. Los diferentes fragmentos. -- 4.2. Los elementos transversales. -- 4.2.1. Gesto melódico. -- 4.2.2. Células. -- 4.2.3. Generación algorítmica de voces melódicas. -- 5. La poliespacialidad sonora. -- 5.1. Concepto poliespacial. -- 5.2. El problema de la notación. -- 5.3. El problema de la realización. -- II. Un universo de armonías al infinito. -- 1. Los espacios sonoros y la generación del timbre. -- 1.1. El piano y el campanario. -- 1.2. El gamelan. -- 1.3. Las kalimbas. -- 2. Campo armónico. -- 3. Los campos armónicos de fragments… -- 3.1. Campo armónico en cuartos de tono. -- 3.2. Campo armónico en séptimos de tono. -- 3.3. Campo armónico del gamelan virtual. -- 3.4. Campos armónicos equipentatónico y equiheptatónico. -- 4. La realización armónica en fragments… -- 5. Tres caminos armónicos posibles en la poliespacialidad. -- 5.1. Armonía propia o paralela. -- 5.2. Armonía sintética o híbrida. -- 5.3. Armonía adaptativa o plástica. -- 5.4. El modelo de fragments… -- Conclusión: Hacia el archipielago… -- Bibliografía. -- Grabaciones. -

Pluralidad y continuidad : espacios sonoros múltiples en la armonía y la estructura de una composición musical

O presente trabalho propõe desvendar uma reflexão sobre a organização em microintervalos da obra fragments pour un labyrinthe de cendres e seus derivados, compostos a partir da ideia de confronto e encontro entre varios espaços sonoros. O processo inclui a avaliação do conceito de espaço sonoro, sua aplicação nas obras e as possibilidades de uma pluralidade de espaços sonoros simultâneos e sucessivos. A geração de uma poliespacialidade sonora coloca o problema da notação, bem como a sua realização concreta. Essa poliespacialidade sonora se manifesta através do uso melódico: suas consequências são analisadas no campo timbrístico dos instrumentos reais e virtuais utilizados na obra, bem como no desenvolvimento de uma harmonia integral estática multifacetada.El presente trabajo se propone desarrollar una reflexión sobre la organización microinterválica de la obra fragments pour un labyrinthe de cendres y sus derivadas, compuestas a partir de la idea de la confrontación y el encuentro entre varios espacios sonoros. El proceso incluye la evaluación del concepto de espacio sonoro, su aplicación en las obras y las posibilidades de una pluralidad de espacios sonoros simultáneos y sucesivos. La generación de una poliespacialidad sonora plantea la problemática notacional así como su realización concreta. Esta poliespacialidad sonora se manifiesta a través del uso melódico: sus consecuencias se analizan en el ámbito tímbrico de los instrumentos reales y virtuales usados en la obra así como en el desarrollo de una armonía integral estática polifacética.The present work intends to develop a reflexion on the microintervalic organization of the work fragments pour un labyrinthe de cendres and its derivatives, composed from the idea of confrontation and encounter between various sound spaces. The process includes the evaluation of the concept of sound space, its application in the works and the possibilities of a plurality of simultaneous and successive sound spaces. The generation of a sound polyspaciality poses the problem of notation as well as its concrete realization. This sound polyspaciality manifests itself through the melodic use: its consequences are analyzed in timbral domain of the real and virtual instruments used in the work as well as in the development of a versatile, static, integral harmony

DNA replication stress-induced loss of reproductive capacity in S. cerevisiae and its inhibition by caloric restriction

In many organisms, attenuation of growth signaling by caloric restriction or mutational inactivation of growth signaling pathways extends lifespan and protects against cancer and other age-related diseases. The focus of many efforts to understand these effects has been on the induction of oxidative stress defenses that inhibit cellular senescence and cell death. Here we show that in the model organism S. cerevisiae, growth signaling induces entry of cells in stationary phase into S phase in parallel with loss of reproductive capacity, which is enhanced by elevated concentrations of glucose. Overexpression of RNR1 encoding a ribonucleotide reductase subunit required for the synthesis of deoxynucleotide triphosphates and DNA replication suppresses the accelerated loss of reproductive capacity of cells cultured in high glucose. The reduced reproductive capacity of these cells is also suppressed by excess threonine, which buffers dNTP pools when ribonucleotide reductase activity is limiting. Caloric restriction or inactivation of the AKT homolog Sch9p inhibits senescence and death in stationary phase cells caused by the DNA replication inhibitor hydroxyurea or by inactivation of the DNA replication and repair proteins Sgs1p or Rad27p. Inhibition of DNA replication stress represents a novel mechanism by which caloric restriction promotes longevity in S. cerevisiae. A similar mechanism may promote longevity and inhibit cancer and other age-related diseases in humans.We wish to thank Molly Burhans for preparing plasmid DNA and Figure 5. This research was supported by a National Cancer Institute Support Grant (P30CA016056) to Roswell Park Cancer Institute and by FCT - Fundacao para a Ciencia e Tecnologia (PTDC/BIA-MIC/114116/2009), Portugal. B. S. M. received a fellowship from FCT (SRFH/BD/41674/2007)

Are SMC complexes loop extruding factors? Linking theory with fact

The extreme length of chromosomal DNA requires organizing mechanisms to both promote functional genetic interactions and ensure faithful chromosome segregation when cells divide. Microscopy and genome‐wide contact frequency analyses indicate that intra‐chromosomal looping of DNA is a primary pathway of chromosomal organization during all stages of the cell cycle. DNA loop extrusion has emerged as a unifying model for how chromosome loops are formed in cis in different genomic contexts and cell cycle stages. The highly conserved family of SMC complexes have been found to be required for DNA cis‐looping and have been suggested to be the enzymatic core of loop extruding machines. Here, the current body of evidence available for the in vivo and in vitro action of SMC complexes is discussed and compared to the predictions made by the loop extrusion model. How SMC complexes may differentially act on chromatin to generate DNA loops and how they could work to generate the dynamic and functionally appropriate organization of DNA in cells is explored

Impact of R-loops on oncogene-induced replication stress in cancer cells

Replication stress is an alteration in the progression of replication forks caused by a variety of events of endogenous or exogenous origin. In precancerous lesions, this stress is exacerbated by the deregulation of oncogenic pathways, which notably disrupts the coordination between replication and transcription, and leads to genetic instability and cancer development. It is now well established that transcription can interfere with genome replication in different ways, such as head-on collisions between polymerases, accumulation of positive DNA supercoils or formation of R-loops. These structures form during transcription when nascent RNA reanneals with DNA behind the RNA polymerase, forming a stable DNA:RNA hybrid. In this review, we discuss how these different cotranscriptional processes disrupt the progression of replication forks and how they contribute to genetic instability in cancer cells

Chromosome Duplication in <i>Saccharomyces cerevisiae</i>

The accurate and complete replication of genomic DNA is essential for all life. In eukaryotic cells, the assembly of the multi-enzyme replisomes that perform replication is divided into stages that occur at distinct phases of the cell cycle. Replicative DNA helicases are loaded around origins of DNA replication exclusively during G 1 phase. The loaded helicases are then activated during S phase and associate with the replicative DNA polymerases and other accessory proteins. The function of the resulting replisomes is monitored by checkpoint proteins that protect arrested replisomes and inhibit new initiation when replication is inhibited. The replisome also coordinates nucleosome disassembly, assembly, and the establishment of sister chromatid cohesion. Finally, when two replisomes converge they are disassembled. Studies in Saccharomyces cerevisiae have led the way in our understanding of these processes. Here, we review our increasingly molecular understanding of these events and their regulation. Keywords: DNA replication; cell cycle; chromatin; chromosome duplication; genome stability; YeastBookNational Institutes of Health (U.S.) (Grant GM-052339

A Multi-Step Pathway for the Establishment of Sister Chromatid Cohesion

The cohesion of sister chromatids is mediated by cohesin, a protein complex containing members of the structural maintenance of chromosome (Smc) family. How cohesins tether sister chromatids is not yet understood. Here, we mutate SMC1, the gene encoding a cohesin subunit of budding yeast, by random insertion dominant negative mutagenesis to generate alleles that are highly informative for cohesin assembly and function. Cohesins mutated in the Hinge or Loop1 regions of Smc1 bind chromatin by a mechanism similar to wild-type cohesin, but fail to enrich at cohesin-associated regions (CARs) and pericentric regions. Hence, the Hinge and Loop1 regions of Smc1 are essential for the specific chromatin binding of cohesin. This specific binding and a subsequent Ctf7/Eco1-dependent step are both required for the establishment of cohesion. We propose that a cohesin or cohesin oligomer tethers the sister chromatids through two chromatin-binding events that are regulated spatially by CAR binding and temporally by Ctf7 activation, to ensure cohesins crosslink only sister chromatids

Chromosome Cohesion: A Cycle of Holding Together and Falling Apart

When a cell prepares to divide, the chromosomes need to separate at just the right moment. Regulating the cohesion of chromosomes is key to achieving thi