Separation of DNA Replication from the Assembly of Break-Competent Meiotic Chromosomes Hannah G. Blitzblau1,2, Clara S. Chan1, Andreas Hochwagen2,3, Stephen P. Bell1* 1 Department of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America, 2 Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, Massachusetts, United States of America, 3 Department of Biology, New York University, New York, New York, United States of America 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. Citation: Blitzblau HG, Chan CS, Hochwagen A, Bell SP (2012) Separation of DNA Replication from the Assembly of Break-Competent Meiotic Chromosomes. PLoS Genet 8(5): e1002643. doi:10.1371/journal.pgen.1002643 Editor: R. Scott Hawley, Stowers Institute for Medical Research, United States of America Received December 20, 2011; Accepted February 17, 2012; Published May 17, 2012 Copyright: 2012 Blitzblau et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: SPB is an investigator of the Howard Hughes Medical Institute. HGB was supported by a predoctoral fellowship from the Howard Hughes Medical Institute. This work was supported by the NIH (GM088248 to AH, www.nigms.nih.gov) and the Howard Hughes Medical Institute (hhmi.org). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: spbell@mit.edu Introduction The meiotic cell division produces haploid gametes from diploid progenitors by segregating the maternally- and paternally-derived copies of each chromosome. The faithful distribution of homol- ogous chromosomes in meiosis is facilitated in most organisms by the crossovers formed during homologous recombination. Meiotic recombination occurs through the carefully orchestrated repair of programmed DNA double-strand breaks (DSBs) and takes place shortly after DNA replication during an extended gap phase referred to as meiotic prophase. Both the formation and faithful repair of meiotic DSBs into crossover recombinants requires the large-scale reorganization of each meiotic chromosome into a series of chromatin loops emanating from a central, condensed axis [1,2]. Pre-meiotic S phase (meiS) is longer than pre-mitotic S phase (mitS) in many organisms [2,3,4], and it has been hypothesized that the protracted DNA synthesis either contributes to, or is affected by, the dramatic chromosome reorganization that occurs during meiotic prophase. The kinetics of genome duplication are determined by where and when DNA replication begins. In eukaryotic genomes, DNA replication initiates from many sites along each chromosome, termed origins of replication, whose likelihood of utilization modulates the length of S phase in different developmental situations [5]. In yeast, potential replication origins are selected during G1 phase by the loading of the Mcm2-7 replicative helicase at specific sites along each chromosome [6,7]. Upon entry into S phase, the activities of cyclin-dependent kinase (CDK) and Dbf4- dependent Cdc7 kinase (DDK) trigger the initiation of DNA replication from a subset of these potential origins [8,9]. The remaining ‘‘inactive’’ origins are passively replicated by forks derived from nearby origins. Studies of individual DNA molecules revealed that the time at which each origin initiates DNA replication during S phase varies substantially between cells, and there is little correlation between distant loci, suggesting origin activation is not coordinated [10,11]. Nevertheless, when the population as a whole is considered, a robust and reproducible replication timing program is seen, regardless of strain background or method used to assess replication timing [8,9,11], suggesting chromosomal DNA replication can be accurately described by a probability function. MeiS in budding yeast has been estimated to last between 1.5–3 times as long as mitS [3,12]. Theoretically, the longer duration of meiS could be due to either reduced efficiency of the initiation of DNA replication (from all or a subset of origins), reduced replication fork rates or a combination of both. Previous studies suggested that the extended length of meiS is not due to changes in origin selection because the majority of the origins on chromo- somes III and VI initiate DNA replication during both mitS and meiS in budding yeast [13,14], and genome-wide analyses PLoS Genetics | www.plosgenetics.org 1 May 2012 | Volume 8 | Issue 5 | e1002643