mitotic cells and Rec8 in meiotic cells [41]. Although the overall levels of binding varied, we found consistent patterns of Hop1, Red1 and Rec8 at CARs in all situations lacking DNA replication examined, indicating that axis formation occurs independently of DNA replication (Figure S7). To determine whether the axes formed in these situations were functional, we measured genome-wide DSB formation by ssDNA enrichment in a cdc6-mn strain (a dmc1D mutation was used to prevent repair of DSBs). We were able to detect DSBs across all chromosomes after 5 hours in sporulation medium (Figure 6C and Figure S8), despite the fact that the genome remained largely unreplicated at this time (Figure 6D). These DSB HSs occurred at the same sites in both dmc1D and cdc6-mn dmc1D cells, although the intensity of DSB formation differed at many sites. Because the FACS analysis indicated that there is some DNA replication occurring in the cdc6-mn strains (Figure 6D, see tailing towards 4C at 5 hours), we were concerned that the ssDNA at DNA replication forks might interfere with the quantitative measure- ment of DSBs in the cdc6-mn cells. Therefore, we measured DSB formation by pulsed-field gel electrophoresis, revealing high levels of DSBs in the cdc6-mn strains (Figure 6E, note that the total signal is lower in the cdc6-mn samples because we normalized for cell number and the chromosomes do not replicate). We conclude that the formation of DSB-competent meiotic chromosomes does not require bulk meiotic DNA replication. Together, our results indicate that pre-meiotic DNA replication and meiotic chromo- some axis assembly are functionally separable processes, and that the formation of a fully DSB-competent chromosome configura- tion can occur in a chromosome-autonomous fashion without the need for a sister chromatid. Discussion We investigated the coordination between pre-meiotic DNA replication and the formation of meiotic chromosome axes. Comparison of replication profiles from meiotic and mitotic cells revealed substantial differences in the regulation of initiation of DNA replication at many origins. Because the majority of replication origins initiated replication later in pre-meiotic cells, we propose that the slower meiS is primarily due to delayed replication initiation. We did not observe a direct link between the formation of DSB-competent chromosome structures and the DNA replication program, indicating that these processes can be functionally separated. Transcription regulates Mcm2-7 loading at a subset of origins Although the Mcm2-7 binding sites were largely the same in both the mitotic and meiotic cell cycles, approximately 9% of sites showed differential Mcm2-7 loading. These sites were much more frequently located within promoters or coding regions of genes, and Mcm2-7 loading appeared to be prevented by gene expression. Previous reports indicated that transcription through an origin is deleterious to replication complex assembly and replication initiation [21,42]. Similar to the situation described here, it has been reported that ARS605 is inactivated by meiosis- specific transcription of the overlapping gene MSH4, which caused the loss of ORC-DNA association [14]. We did not identify ARS605 as a mitosis-specific origin in this study (Figure S1), possibly because we collected samples for Mcm2-7 analysis relatively early in the meiotic cell cycle, when MSH4 transcription was not yet fully activated and residual amounts of Mcm2-7 were still bound to the DNA. Alternatively, the low levels of Mcm2-7 we detected at ARS605 are insufficient for initiation. However, we Separating DNA Synthesis and Meiotic DSB Formation Figure 6. DNA replication is not required for axis association or DSB formation. (A) Indirect immunofluorescence for Red1 (green) and DAPI staining for total DNA (blue) on spread nuclei from cells at 3 hours after inoculation into SPO for wild-type cells (H119) with and without HU, cdc6-mn cells (H154) and clb5D clb6D cells (H2017). (B) Hop1 localization analysis was performed for wild-type cells with (H4471) and without HU (H119, [67]), cdc6-mn cells (H154) and clb5D clb6D cells (H2017). The enrichment of Hop1 over input DNA is plotted for chromosome III. (C) ssDNA enrichment in dmc1D cells (H118, [67]), and dmc1D cdc6-mn cells (H1584) was plotted with respect to position on chromosome VII. (D) FACS analysis was performed for dmc1D (H118) and dmc1D cdc6-mn cells (H1584). (E) CHEF gel analysis of chromosome VIII during a meiotic time course using dmc1D (H118) and dmc1D cdc6-mn cells (H4534). doi:10.1371/journal.pgen.1002643.g006 PLoS Genetics | www.plosgenetics.org 8 May 2012 | Volume 8 | Issue 5 | e1002643