Effect of Chromosome Tethering on Nuclear Organization in Yeast

Avşaroğlu, Barış; Bronk, Gabriel; Gordon-Messer, Susannah; Ham, Jungoh; Bressan, Debra A.; Haber, James E.; Kondev, Jané

doi:10.1371/journal.pone.0102474

Cited by 29 publications

(43 citation statements)

References 78 publications

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“…The can1 recipient locus lies only 33 kb from a telomere, and a DSB at this site might be constrained in its homology search, although we found previously that sites >20 kb from telomeres should not be confined by telomere tethering (13). To be certain that the strong correlation in repair locations was not caused by the location of the DSB at can1, we created eight additional single-donor strains in which the leu2::HOcs was inserted 200 kb from its telomere on Chr 2 (the location of the reference donor in the competition assay described above) (Fig.…”

Section: Contact Frequency Is Also Highly Correlated With Use In a Comentioning

confidence: 88%

Chromosome position determines the success of double-strand break repair

Lee

Wang

Chang

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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119

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Repair of a chromosomal double-strand break (DSB) by gene conversion depends on the ability of the broken ends to encounter a donor sequence. To understand how chromosomal location of a target sequence affects DSB repair, we took advantage of genome-wide Hi-C analysis of yeast chromosomes to create a series of strains in which an induced site-specific DSB in budding yeast is repaired by a 2-kb donor sequence inserted at different locations. The efficiency of repair, measured by cell viability or competition between each donor and a reference site, showed a strong correlation (r = 0.85 and 0.79) with the contact frequencies of each donor with the DSB repair site. Repair efficiency depends on the distance between donor and recipient rather than any intrinsic limitation of a particular donor site. These results further demonstrate that the search for homology is the rate-limiting step in DSB repair and suggest that cells often fail to repair a DSB because they cannot locate a donor before other, apparently lethal, processes arise. The repair efficiency of a donor locus can be improved by four factors: slower 5′ to 3′ resection of the DSB ends, increased abundance of replication protein factor A (RPA), longer shared homology, or presence of a recombination enhancer element adjacent to a donor.homologous recombination | double-strand break repair | chromosome conformation | homology search | donor location H omologous recombination is the predominant mechanism to repair chromosome breaks and preserve genome integrity. In eukaryotes, the broken double-strand break (DSB) ends undergo extensive 5′ to 3′ resection, promoting the binding of the Rad51 recombinase to form a nucleoprotein filament that can search the genome for a homologous sequence with which it can effect repair. Donor template sequences can be located on a sister chromatid, a homologous chromosome or an ectopic location. When ectopic sequences are used, repair results in nonallelic replacements of sequences. In budding yeast Saccharomyces cerevisiae it is possible to monitor the sequence of DSB repair events in real time by Southern blots, PCR or chromatin immunoprecipitation (1).Haploid yeast chromosomes are arranged in a Rabl orientation, with the 16 centromeres all clustered at the spindle-pole body (SPB) whereas the telomeres are associated in loose clusters at the nuclear envelope (2). These observations have been extended by the use of chromosome conformation capture approaches (3, 4) to map the relative positions of loci along each chromosome based on their frequencies of crosslinking (contact frequencies) with many other sites in the genome. Previous studies have shown that telomere-associated sequences preferentially recombine with other telomere-associated loci whereas centromere-linked sites selectively recombine with other centromere-linked loci (5-7). However, such preferences, presumably caused by the constraints of tethering, may not reflect the general behavior of most sequences undergoing homologous recombination. It is not known how the ...

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Section: Contact Frequency Is Also Highly Correlated With Use In a Comentioning

confidence: 88%

Chromosome position determines the success of double-strand break repair

Lee

Wang

Chang

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

119

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“…1B account for the loss of entropy due to chromosome folding, and they are readily computed using the polymer model of Chr III illustrated in Fig. 1C (23). The thermodynamic weights are nothing but the relative probabilities of a contact occurring between the different genetic loci involved in mating-type switching, as illustrated in the first column of Fig.…”

Section: Avşaroglu Et Almentioning

confidence: 99%

“…To quantitatively test predictions based on the chromosomerefolding model we made use of the wild-type yeast strain with a 256-copy tandem array of LacO sequences, which bind LacI-GFP, inserted 1.5 kb proximal to the HML locus, and a 112-copy tandem array of TetO sequences, which bind TetR-RFP, inserted 5 kb distal to the MAT locus ( Fig. 2A) (23,(55)(56)(57).…”

Section: Avşaroglu Et Almentioning

confidence: 99%

“…We then collected cells at different time points (t) after inducing the DSB, fixed them with paraformaldehyde, and imaged the fixed yeast cells using confocal microscopy. The images were analyzed using the ImageJ plugin SpotDistance to measure the 3D distances (d) between fluorescent markers at MAT and HML (23,58). Details of quantitative fluorescence microscopy are explained in Materials and Methods.…”

Section: Avşaroglu Et Almentioning

confidence: 99%

“…Namely, the chromosomes' spatial organization is described in quantitative detail by a simple model of chromosomes as random-walk polymers confined to a spherical volume, which represents the nucleus (20)(21)(22)(23)(24)(25)(26)(27)(28)(29). Another element of organization is the tethering of chromosomes to the nuclear envelope at their telomeres and to the spindle pole body at their centromeres (2,22).…”

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Chromosome-refolding model of mating-type switching in yeast

Avşaroğlu

Bronk

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Chromosomes are folded into cells in a nonrandom fashion, with particular genetic loci occupying distinct spatial regions. This observation raises the question of whether the spatial organization of a chromosome governs its functions, such as recombination or transcription. We consider this general question in the specific context of mating-type switching in budding yeast, which is a model system for homologous recombination. Mating-type switching is induced by a DNA double-strand break (DSB) at the MAT locus on chromosome III, followed by homologous recombination between the cut MAT locus and one of two donor loci (HMLα and HMRa), located on the same chromosome. Previous studies have suggested that in MATa cells after the DSB is induced chromosome III undergoes refolding, which directs the MAT locus to recombine with HMLα. Here, we propose a quantitative model of mating-type switching predicated on the assumption of DSB-induced chromosome refolding, which also takes into account the previously measured stochastic dynamics and polymer nature of yeast chromosomes. Using quantitative fluorescence microscopy, we measure changes in the distance between the donor (HMLα) and MAT loci after the DSB and find agreement with the theory. Predictions of the theory also agree with measurements of changes in the use of HMLα as the donor, when we perturb the refolding of chromosome III. These results establish refolding of yeast chromosome III as a key driving force in MAT switching and provide an example of a cell regulating the spatial organization of its chromosome so as to direct homology search during recombination.chromosome organization | homologous recombination | random-walk polymers | quantitative fluorescent microscopy | statistical physics C hromosomes in bacteria and eukaryotic nuclei are folded in a nonrandom fashion, which leads to specific DNA sequences assuming narrowly distributed positions within the cell or the nucleus (1-4). An interesting question is, then, to what extent does this spatial organization of chromosomes determine their function? For example, differential gene expression during development and the maintenance of genomic integrity in response to damaging agents have both been linked to chromosome organization. In both cases the folded state of chromosomes determines which functionally related DNA sequences are spatially close to each other, which in turn enables their function (5-11). Furthermore, earlier theoretical studies addressing aspects of long-distance regulatory interactions between DNA sequences have suggested that formation of chromosomal loops can be used to modulate the frequency of these interactions (12, 13). Double-strand breaks (DSBs) are deleterious DNA lesions that can be repaired by homologous recombination. The repair process requires spatial proximity, of about 10 nm or so, of the region around the DSB and a homologous DNA sequence, which is located either on the same or on a different chromosome (14-17). Recent studies have revealed a role for the spatial organization of...

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Promoting convergence: The integrated graduate program in physical and engineering biology at Yale University, a new model for graduate education

Noble

Mochrie

O’Hern

et al. 2016

Biochem Molecular Bio Educ

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In 2008, we established the Integrated Graduate Program in Physical and Engineering Biology (IGPPEB) at Yale University. Our goal was to create a comprehensive graduate program to train a new generation of scientists who possess a sophisticated understanding of biology and who are capable of applying physical and quantitative methodologies to solve biological problems. Here we describe the framework of the training program, report on its effectiveness, and also share the insights we gained during its development and implementation. The program features co‐teaching by faculty with complementary specializations, student peer learning, and novel hands‐on courses that facilitate the seamless blending of interdisciplinary research and teaching. It also incorporates enrichment activities to improve communication skills, engage students in science outreach, and foster a cohesive program cohort, all of which promote the development of transferable skills applicable in a variety of careers. The curriculum of the graduate program is integrated with the curricular requirements of several Ph.D.‐granting home programs in the physical, engineering, and biological sciences. Moreover, the wide‐ranging recruiting activities of the IGPPEB serve to enhance the quality and diversity of students entering graduate school at Yale. We also discuss some of the challenges we encountered in establishing and optimizing the program, and describe the institution‐level changes that were catalyzed by the introduction of the new graduate program. The goal of this article is to serve as both an inspiration and as a practical “how to” manual for those who seek to establish similar programs at their own institutions. © 2016 by The International Union of Biochemistry and Molecular Biology, 44(6):537–549, 2016.

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Effect of Chromosome Tethering on Nuclear Organization in Yeast

Cited by 29 publications

References 78 publications

Chromosome position determines the success of double-strand break repair

Chromosome position determines the success of double-strand break repair

Chromosome-refolding model of mating-type switching in yeast

Promoting convergence: The integrated graduate program in physical and engineering biology at Yale University, a new model for graduate education

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