Analysis of the Chromosomal Localization of Yeast SMC Complexes by Chromatin Immunoprecipitation

Makrantoni, Vasso; Robertson, Daniel; Marston, Adèle L.

doi:10.1007/978-1-4939-9520-2_10

Cited by 8 publications

(8 citation statements)

References 24 publications

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“…We followed the protocol of calibrated ChIP [79] ) and further lysed by bead beating (BioSpec Products) with 0.5-mm glass beads (Biospec Products). To shear chromatin, a Covaris S220 instrument was used with the following program: peak incident power, 175; duty factor, 10%; cycle per burst, 200; treatment time, 250 seconds.…”

Section: Chip and Qpcrmentioning

confidence: 99%

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Evolutionary repair: Changes in multiple functional modules allow meiotic cohesin to support mitosis

et al. 2020

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The role of proteins often changes during evolution, but we do not know how cells adapt when a protein is asked to participate in a different biological function. We forced the budding yeast, Saccharomyces cerevisiae, to use the meiosis-specific kleisin, recombination 8 (Rec8), during the mitotic cell cycle, instead of its paralog, Scc1. This perturbation impairs sister chromosome linkage, advances the timing of genome replication, and reduces reproductive fitness by 45%. We evolved 15 parallel populations for 1,750 generations, substantially increasing their fitness, and analyzed the genotypes and phenotypes of the evolved cells. Only one population contained a mutation in Rec8, but many populations had mutations in the transcriptional mediator complex, cohesin-related genes, and cell cycle regulators that induce S phase. These mutations improve sister chromosome cohesion and delay genome replication in Rec8-expressing cells. We conclude that changes in known and novel partners allow cells to use an existing protein to participate in new biological functions.

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Section: Chip and Qpcrmentioning

confidence: 99%

“…For ChIP-Seq, chromatin was immunoprecipitated as described above, and purified chromatin was subjected to DNA end repair and dA tailing to make the sequencing library as described [79]. Samples were sequenced on a MiniSeq with 75 base paired-end reads (Illumina, San Diego, CA).…”

Section: Chip and Qpcrmentioning

confidence: 99%

Evolutionary repair: Changes in multiple functional modules allow meiotic cohesin to support mitosis

et al. 2020

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“…ChIP-seq experiments used a protocol that combined and modified previous methods ((MURAKAMI AND KEENEY 2014;MAKRANTONI et al 2019); Hajime Murakami, personal communication). Strains used contained the URA3-tel-ARG4-parS reporter construct inserted at URA3.…”

Section: Calibrated Chromatin Immunoprecipitation and Sequencing (Chi...mentioning

confidence: 99%

“…ChIP-seq data were calibrated as described (MAKRANTONI et al 2019). Briefly, single ended fastq format sequences derived from ChIP-seq data were quality-trimmed using fastp (CHEN et al 2018).…”

Section: Calibrated Chromatin Immunoprecipitation and Sequencing (Chi...mentioning

confidence: 99%

Turning coldspots into hotspots: targeted recruitment of axis protein Hop1 stimulates meiotic recombination inSaccharomyces cerevisiae

Shodhan

Xaver

Wheeler

et al. 2022

Preprint

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The DNA double strand breaks (DSBs) that initiate meiotic recombination are formed in the context of the meiotic chromosome axis, which in budding yeast contains a meiosis-specific cohesin isoform and the meiosis-specific proteins Hop1 and Red1. Hop1 and Red are important for DSB formation; DSB levels are reduced in their absence and their levels, which vary along the lengths of chromosomes, are positively correlated with DSB levels. How axis protein levels influence DSB formation and recombination remains unclear. To address this question, we developed a novel approach that uses a bacterial ParB-parS partition system to recruit axis proteins at high levels to inserts at recombination coldspots where Hop1 and Red1 levels are normally low. Recruiting Hop1 markedly increased DSBs and homologous recombination at target loci, to levels equivalent to those observed at endogenous recombination hotspots. This local increase in DSBs did not require Red1 or the meiosis-specific cohesin component Rec8, indicating that, of the axis proteins, Hop1 is sufficient to promote DSB formation. However, while most crossovers at endogenous recombination hotspots are formed by the meiosis-specific MutLγ resolvase, only a small fraction of crossovers that formed at an insert locus required MutLγ, regardless of whether or not Hop1 was recruited to that locus. Thus, while local Hop1 levels determine local DSB levels, the recombination pathways that repair these breaks can be determined by other factors, raising the intriguing possibility that different recombination pathways operate in different parts of the genome.

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“…We used the following formula: For ChIP-Seq, chromatin was immunoprecipitated as described above, and purified 707 chromatin was subjected to DNA end repair and dA tailing to make sequencing library as described 708 (Makrantoni et al, 2019). Samples were sequenced on a MiniSeq with 75 base paired-end reads 709 (Illumina, San Diego, CA).…”

mentioning

confidence: 99%

Evolutionary repair: changes in multiple functional modules allow meiotic cohesin to support mitosis

Hsieh

Makrantoni

Robertson

et al. 2019

Preprint

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Different members of the same protein family often perform distinct cellular functions. How much are these differing functions due to changes in a protein’s biochemical activity versus changes in other proteins? We asked how the budding yeast, Saccharomyces cerevisiae, evolves when forced to use the meiosis-specific kleisin, Rec8, instead of the mitotic kleisin, Scc1, during the mitotic cell cycle. This perturbation impairs sister chromosome linkage and reduces reproductive fitness by 45%. We evolved 15 populations for 1750 generations, substantially increasing their fitness, and analyzed their genotypes and phenotypes. We found no mutations in Rec8, but many populations had mutations in the transcriptional mediator complex, cohesin-related genes, and cell cycle regulators that induce S phase. These mutations improve sister chromosome cohesion and slow genome replication in Rec8-expressing cells. We conclude that changes in known and novel partners allow proteins to improve their ability to perform new functions.

show abstract

Analysis of the Chromosomal Localization of Yeast SMC Complexes by Chromatin Immunoprecipitation

Cited by 8 publications

References 24 publications

Evolutionary repair: Changes in multiple functional modules allow meiotic cohesin to support mitosis

Evolutionary repair: Changes in multiple functional modules allow meiotic cohesin to support mitosis

Turning coldspots into hotspots: targeted recruitment of axis protein Hop1 stimulates meiotic recombination inSaccharomyces cerevisiae

Evolutionary repair: changes in multiple functional modules allow meiotic cohesin to support mitosis

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