A chemostat array enables the spatio-temporal analysis of the yeast proteome

Dénervaud, Nicolas; Becker, Johannes; Delgado-Gonzalo, Ricard; Damay, Pascal; Rajkumar, Arun S.; Unser, Michaël; Shore, David; Naef, Félix; Maerkl, Sebastian J.

doi:10.1073/pnas.1308265110

Cited by 124 publications

(156 citation statements)

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“…Proteins that localize in nuclear foci have been identified in candidate approaches (Lisby et al 2001Melo et al 2001;Zhu et al 2008;Germann et al 2011) and in genome-scale screens (Tkach et al 2012;Denervaud et al 2013;Mazumder et al 2013;Yu et al 2013). Nuclear foci are commonly thought of as centers of DNA repair, in part because foci formed by recombination repair proteins colocalize with double-strand DNA breaks (Lisby et al 2003).…”

Section: Resultsmentioning

confidence: 99%

MTE1 Functions with MPH1 in Double-Strand Break Repair

et al. 2016

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Double-strand DNA breaks occur upon exposure of cells to ionizing radiation and certain chemical agents or indirectly through replication fork collapse at DNA damage sites. If left unrepaired, double-strand breaks can cause genome instability and cell death, and their repair can result in loss of heterozygosity. In response to DNA damage, proteins involved in double-strand break repair by homologous recombination relocalize into discrete nuclear foci. We identified 29 proteins that colocalize with recombination repair protein Rad52 in response to DNA damage. Of particular interest, Ygr042w/Mte1, a protein of unknown function, showed robust colocalization with Rad52. Mte1 foci fail to form when the DNA helicase gene MPH1 is absent. Mte1 and Mph1 form a complex and are recruited to double-strand breaks in vivo in a mutually dependent manner. MTE1 is important for resolution of Rad52 foci during double-strand break repair and for suppressing break-induced replication. Together our data indicate that Mte1 functions with Mph1 in double-strand break repair.KEYWORDS DNA repair; recombination; double-strand breaks; break-induced replication; loss of heterozygosity; nuclear foci E FFECTIVE repair of double-strand DNA breaks (DSBs) is critical to the preservation of genome stability, yet most modes of DSB repair have significant potential to generate sequence alterations or sequence loss. Repair of DSBs by homologous recombination can result in loss of heterozygosity when resolution of recombination intermediates between homologous chromosomes results in a crossover. As such, cells possess several mechanisms by which crossing over can be suppressed in favor of noncrossover recombination products. Double Holliday junction (dHJ) intermediates that result from invasion of a homologous chromosome by both ends of a resected DSB (Szostak et al. 1983) can be resolved nucleolytically by the action of the Yen1 and Mus81/Mms4 endonucleases (Blanco et al. 2010;Ho et al. 2010) to produce a random distribution of crossover and noncrossover products. By contrast, the same dHJ intermediates can be dissolved by the combined helicase and ssDNA decatenase action of the Bloom/TopIIIa/Rmi1 complex (Sgs1/Top3/Rmi1 in yeast) (Wu et al. 2006;Yang et al. 2010) to yield exclusively noncrossover products (Wu and Hickson 2003). Crossovers can also be prevented if the D-loop structure that results from the first strand invasion by one end of a resected DSB into the homologous chromosome is unwound before capture of the second end to form the dHJ. Unwinding of D-loops is catalyzed in vitro and in vivo by the 39-to-59 DNA helicase Mph1 (Sun et al. 2008;Prakash et al. 2009) to prevent loss of heterozygosity due to crossovers and break-induced replication (BIR) (Luke-Glaser and Luke 2012;Mazon and Symington 2013;Stafa et al. 2014).The Mph1 DNA helicase was first identified as a deletion mutant with an increased mutation frequency (Entian et al. 1999). Subsequent characterization revealed that mph1 mutants are sensitive to the alkylating agent M...

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Section: Resultsmentioning

confidence: 99%

MTE1 Functions with MPH1 in Double-Strand Break Repair

et al. 2016

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“…Commercial fermenters that have been modified for chemostat use, such as those from New Brunswick or Infors, typically range from 50 mL to 2 L. Custom glass-blown or otherwise home-built devices typically fall toward the lower end of this range and below (e.g., Klein et al 2013). Microfluidic implementations weigh in at just a few microliters (Cookson et al 2005;Groisman et al 2005;Dénervaud et al 2013). There are several important considerations to take into account when determining which system scale is most appropriate.…”

Section: Chemostat Designmentioning

confidence: 99%

Chemostat Culture for Yeast Physiology and Experimental Evolution

Dunham

Kerr

Miller

et al. 2017

Cold Spring Harb Protoc

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Continuous culture provides many benefits over the classical batch style of growing yeast cells. Steadystate cultures allow for precise control of growth rate and environment. Cultures can be propagated for weeks or months in these controlled environments, which is important for the study of experimental evolution. Despite these advantages, chemostats have not become a highly used system, in large part because of their historical impracticalities, including low throughput, large footprint, systematic complexity, commercial unavailability, high cost, and insufficient protocol availability. However, we have developed methods for building a relatively simple, low-cost, small footprint array of chemostats that can be run in multiples of 32. This "ministat array" can be applied to problems in yeast physiology and experimental evolution. BATCH CULTUREThe most common method of growing yeast cell cultures is the standard "batch" culture. In this regime, a small number of cells are inoculated into nutrient-rich medium and allowed to divide at maximal growth rate through nutrient exhaustion and into saturation. Sampling of the culture for the experiment of choice is most typically done from the saturated culture or from cells growing in midlog phase before the diauxic shift. Batch culture provides a number of obvious benefits-it is easy, uses glassware commonly available in any laboratory, and is consistent with a vast literature. However, batch cultures can be problematic for certain applications. When comparing cells of different genotypes, for example, differences in the maximal growth rate are a common phenotype. Many other phenotypes co-vary with growth rate, leading to a nonspecific suite of cell biological, gene expression, and other physiological changes that may not be directly related to the mutation of interest (Regenberg et al. 2006;Castrillo et al. 2007;Brauer et al. 2008).Batch culture has also been a standard for long-term evolution experiments. Depending on the question being asked, batch culture with serial dilution can be a simple and scalable solution (e.g., Zeyl et al. 2003;Lang et al. 2011). However, such cultures are only rarely propagated in a relatively constant environment and growth rate regime because doing so can require a heroic sampling regimen where back dilution is performed multiple times per day (e.g., Torres et al. 2010). More typically, cultures are allowed to exhaust nutrients before transfer to new medium, leading to a variation in growth rate and nutrient access over the evolutionary time course. These discontinuities in selective pressure can lead to complex subpopulation structures in which different genotypes specialize for the various stages of the growth cycle (e.g., dividing a few extra times or failing to die after saturation, shortening lag phase,

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“…Microbial populations will grow in proportion to this rate. Contemporary versions of this device have been scaled downwards to achieve larger numbers of experimental replicates or to the point of single cell analysis (Dénervaud et al 2013). Discoveries made using chemostats are rooted in physiological responses to environmental stresses.…”

Section: Technologies Relevant To Performing Experimental Microbial Ementioning

confidence: 99%