Biology subjects

Koszul, R.

Publications and source records attributed to Koszul, R..

4 recordsLinked to original sources

bioRxiv2018

Dynamic Processing of Displacement Loops During Recombinational DNA Repair

Displacement-loops (D-loops) are pivotal intermediates of homologous recombination (HR), a universal DNA double strand break (DSB) repair pathway. We developed a versatile assay for the physical detection of D-loops in vivo, which enabled studying the kinetics of their formation and defining the network of D-loop formation and reversal pathways. Nascent D-loops are detected within 2 hrs of DSB formation and extended over the next 2 hrs in a system allowing break-induced replication. The majority of D-loops are disrupted in wild type cells by two pathways: one supported by the Srs2 helicase and the other by the Mph1 helicase and the Sgs1-Top3-Rmi1 helicase-topoisomerase complex. Both pathways operate without significant overlap and are delineated by the Rad54 paralog Rdh54 in an ATPase-independent fashion. This study uncovers a novel layer of HR control in cells relying on nascent D-loop dynamics, revealing unsuspected complexities, and identifying a surprising role for a conserved Rad54 paralog.

molecular biology↗

bioRxiv2018

Synthetic chromosome fusion: effects on genome structure and function

As part of the Synthetic Yeast 2.0 (Sc2.0) project, we designed and synthesized synthetic chromosome I. The total length of synI is [~]21.4% shorter than wild-type chromosome I, the smallest chromosome in Saccharomyces cerevisiae. SynI was designed for attachment to another synthetic chromosome due to concerns of potential instability and karyotype imbalance. We used a variation of a previously developed, robust CRISPR-Cas9 method to fuse chromosome I to other chromosome arms of varying length: chrIXR (84kb), chrIIIR (202kb) and chrIVR (1Mb). All fusion chromosome strains grew like wild-type so we decided to attach synI to synIII. Through the investigation of three-dimensional structures of fusion chromosome strains, unexpected loops and twisted structures were formed in chrIII-I and chrIX-III-I fusion chromosomes, which depend on silencing protein Sir3. These results suggest a previously unappreciated 3D interaction between HMR and the adjacent telomere. We used these fusion chromosomes to show that axial element Red1 binding in meiosis is not strictly chromosome size dependent even though Red1 binding is enriched on the three smallest chromosomes in wild-type yeast, and we discovered an unexpected role for centromeres in Red1 binding patterns.

synthetic biology↗

bioRxiv2017

Redesigning chromosomes to optimize conformation capture (Hi-C) assays

In all chromosome conformation capture based experiments the accuracy with which contacts are detected varies considerably because of the uneven distribution of restriction sites along genomes. In addition, repeated sequences as well as homologous, large identical regions remain invisible to the assay because of the ambiguities they introduce during the alignment of the sequencing reads along the genome. As a result, the investigation of homologs during meiosis prophase through 3C studies has been limited. Here, we redesigned and reassembled in yeast a 145kb region with regularly spaced restriction sites for various enzymes. Thanks to this Syn-3C design, we enhanced the signal to noise ratio and improved the visibility of the entire region. We also improved our understanding of Hi-C data and definition of resolution. The redesigned sequence is now distinguishable from its native homologous counterpart in an isogenic diploid strain. As a proof of principle, we track the establishment of homolog pairing during meiotic prophase in a synchronized population. This provides new insights on the individualization and pairing of homologs, as well as on their internal restructuration into arrays of loops during meiosis prophase. Overall, we show the interest of redesigned genomic regions to explore complex biological questions otherwise difficult to address.

synthetic biology↗

bioRxiv2016

Choreography of budding yeast chromosomes during the cell cycle

To ensure the proper transmission of the genetic information, DNA molecules must be faithfully duplicated and segregated. These processes involve dynamic modifications of chromosomes internal structure to promote their individualization, as well as their global repositioning into daughter cells (Guacci et al., 1994; Kleckner et al., 2014; Mizuguchi et al., 2014). In eukaryotes, these events are regulated by conserved architectural proteins, such as structural maintenance of chromosomes (SMC i.e. cohesin and condensin) complexes (Aragon et al., 2013a; Uhlmann, 2016). Although the roles of these factors have been actively investigated, the genome-wide chromosomal architecture and dynamics both at small and large-scales during cell division remains elusive. Here we report a comprehensive Hi-C (Dekker et al., 2002; Lieberman-Aiden et al., 2009) analysis of the dynamic changes of chromosomes structure over the Saccharomyces cerevisiae cell cycle. We uncover specific SMC-dependent structural transitions between the different phases of the mitotic cycle. During replication, cohesion establishment promotes the increase of long-range intra-chromosomal contacts. This process correlates with the individualization of chromosomes, which culminates at metaphase. Mitotic chromosomes are then abruptly reorganized in anaphase by the mechanical forces exerted by the mitotic spindle on the centromere cluster. The formation of a condensin-dependent loop, that bridges centromere cluster with the cenproximal flanking region of the rDNA, suggests that these forces may directly facilitate nucleolus segregation. This work provides a comprehensive overview of chromosome dynamics during the cell cycle of a unicellular eukaryote that recapitulates and unveils new features of highly conserved stages of the cell division.

genetics↗