Separation of DNA Replication from the Assembly of Break-Competent Meiotic Chromosomes

Abstract

The meiotic cell division reduces the chromosome number from diploid to haploid to form gametes for sexual reproduction. Although much progress has been made in understanding meiotic recombination and the two meiotic divisions, the processes leading up to recombination, including the prolonged pre-meiotic S phase (meiS) and the assembly of meiotic chromosome axes, remain poorly defined. We have used genome-wide approaches in Saccharomyces cerevisiae to measure the kinetics of pre-meiotic DNA replication and to investigate the interdependencies between replication and axis formation. We found that replication initiation was delayed for a large number of origins in meiS compared to mitosis and that meiotic cells were far more sensitive to replication inhibition, most likely due to the starvation conditions required for meiotic induction. Moreover, replication initiation was delayed even in the absence of chromosome axes, indicating replication timing is independent of the process of axis assembly. Finally, we found that cells were able to install axis components and initiate recombination on unreplicated DNA. Thus, although pre-meiotic DNA replication and meiotic chromosome axis formation occur concurrently, they are not strictly coupled. The functional separation of these processes reveals a modular method of building meiotic chromosomes and predicts that any crosstalk between these modules must occur through superimposed regulatory mechanisms.

Local chromosome context is a major determinant of crossover pathway biochemistry during budding yeast meiosis

“…1/50th of the sample (10 ml) was removed as input, and 2 ml of antiHop1 (a generous gift from Nancy Hollingsworth) was added to the remainder (~490 ml) and incubated with gentle agitation overnight at 4˚C. Antibody complexes were purified by addition of 20 ml of 50% slurry of Gammabind G Sepharose beads (GE Healthcare 17088501), with further incubation for 3 hr at 4˚C, followed by pelleting at 845 RCF for 30 s. Beads were then processed for DNA extraction (Blitzblau et al, 2012;Viji Subramanian and Andreas Hochwagen, personal communication). Beads were washed with 1 ml lysis Buffer and once each with 1 ml high salt lysis buffer (same as lysis buffer except with 500 mM NaCl), 1 ml ChIP wash buffer (10 mM Tris, 0.25M LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA) and 1 mL 10 mM Tris, 1 mM EDTA; all washes were done for 5 min at room temperature.…”

Behavior of replication origins in Eukaryota – spatio-temporal dynamics of licensing and firing

“…Transcription directly through RO disrupts the origin by interference with ORC and MCM binding, [92][93] so some changes in transcription can also cause alterations in origin competence (either positive or negative). Nevertheless, no changes in ORC and/or MCM binding (level-and timedependent) were observed in cells with deregulated timing mechanisms in fkh1D, fkh2D, rif1D, taz1D yeast or Rif1-depleted HeLa cells.…”

The Double-Strand Break Landscape of Meiotic Chromosomes Is Shaped by the Paf1 Transcription Elongation Complex in Saccharomyces cerevisiae

“…Relative replication timing of hotspot classes was determined using published replication timing data of wild-type cells (Blitzblau et al 2012; GEO accession no. GSE35667).…”

“…These data were then used to calculate average nucleosome occupancy as a function of distance from the hotspot summits. Relative replication timing of hotspot classes was determined using published replication timing data of wild-type cells (Blitzblau et al 2012; GEO accession no. GSE35667).…”