identified additional origins, at which Mcm2-7 association was similarly regulated by transcription of the locus. We note that none of the meiosis-specific origins identified in this study were novel; either ARS activity or Mcm2-7 binding was detected in previous studies using mitotically dividing cells (http://www.oridb.org). However, none of them were shown to initiate DNA replication in genome-wide mitotic studies, suggesting they do not function in their chromosomal context. Because these transcriptionally- regulated origins were located close to other origins, their inactivation is unlikely to have a substantial effect on the completion of DNA replication. Delayed DNA replication during meiS Given that origin selection and activation were highly similar in meiS and mitS, the reduced rate of meiS must be due to delayed replication initiation or slow fork progression rates. The presence of a greater amount of noise in the meiS replication profiles made it impossible to measure relative fork rates in meiS and mitS, as we could not create an algorithm to specifically locate and measure all fork progression regions. Despite this difficulty, we noted that in some regions of the genome the scaled meiS and mitS profiles had similar slopes (Figure S2). Because the time of meiS is approximately twice the length of mitS and the profiles are scaled to S phase length, a similar slope indicates that the meiotic fork rates are approximately half the rate of the mitotic forks. On the other hand, we found strong evidence for a delay in replication initiation, as the majority of origins replicated later in meiS than mitS. This replication delay could be due to later or reduced efficiency of initiation, which the CGH method does not distinguish. In support of delayed initiation, a much smaller number of meiS origins initiated replication in HU. Although previous studies observed a similar efficiency of origin usage in meiS and mitS [13,14], we found that the small chromosomes monitored previously showed smaller delays in meiS replication than larger chromosomes (Figure S2), leaving open the possibility that many origins exhibit a reduced efficiency of initiation in meiS. Recent studies suggest that timing and efficiency are linked [43,44], so it is possible that both contribute to the delayed replication in meiS. We postulate that the reduced replication initiation in meiS is caused by a limiting initiation factor. One candidate is the level of CDK activity, which is required for replication initiation. Removal of the cyclin Clb5 in mitotic cells causes a reduction in late origin activation, but the presence of Clb6/CDK substitutes to drive mitS [45,46]. In meiotic cells, CLB5 deletion has an even more severe phenotype, with very delayed and inefficient replication [37], suggesting residual Clb6/CDK activity is lower in meiotic cells. It seems less likely that DDK activity is limiting in meiS, as the use of an analog-sensitive allele of Cdc7 revealed that pre- meiotic DNA replication is virtually unaffected in the presence of the inhibitor [47]. It has been proposed that DDK must act at each individual origin at the time of initiation of DNA replication [48,49], and it is possible that because replication initiation is spread out over a much longer time period in meiS, less DDK activity is required to support these lower initiation rates. A second and non-exclusive model is that slow pre-meiotic DNA replication is caused by the reduced dNTP levels in meiS [50], which presumably occur because of the starvation conditions used to induce sporulation. Lowered nucleotide levels could account for a decrease in both initiation and fork progression rates. This hypothesis is reminiscent of studies of mitotic growth in the presence of HU, which also causes both a delay in origin activation and slower fork rates, resulting in a protracted S phase [27]. The Separating DNA Synthesis and Meiotic DSB Formation observation that meiotic cells arrest more tightly in response to HU is also consistent with a model of reduced dNTP levels. Why is replication initiation delayed in meiS? The generalized delay in replication initiation observed in meiS is very similar to the scaling of S phase observed in other mutants that slow DNA replication [25], suggesting that cells respond to S phase challenges by decreasing the number of active forks. Although a slow S phase would be detrimental in competitive mitotic cultures because it would decrease growth rate, the meiotic program is a form of terminal differentiation and cells are not prepared to divide again immediately following meiotic exit. In sporulating cells, it may be advantageous to proceed slowly but accurately through the cell cycle. It has recently been shown that excessive replication initiation leads to genome instability [43], and meiotic errors would be propagated by the progeny. In higher eukaryotes, meiosis takes place only in germ cells, which differentiate within special organs in response to specific developmental cues. Although these germ cells are not limited by nutrient availability, they may also be optimized for fidelity. Early replication of centromere-proximal origins It is interesting to note that not all origins were equally delayed during meiS; centromere-proximal origins initiated replication efficiently during both meiS (This study and [13]). The conservation of early replication timing of centromeres in meiS and mitS (reviewed in [33], as well as in distantly related yeast species [34], suggests that these sites play a critical role in determining the replication timing program. Consistent with this idea, it has been observed that moving a centromere was sufficient to change replication timing of adjacent origin sequences [51]. One hypothesis for early centromere replication is that it is important for kinetochore function, because mutants that change replication timing were shown to interfere with chromosome segregation [52]. Given that every chromosome has a centromere, it is also possible that linking early replication to centromeres helps to guarantee that every chromosome initiates replication in S phase, even when replication is compromised. Separation of pre-meiotic DNA replication from the assembly of break-competent meiotic chromosomes Although it was previously observed that Rec8 and Spo11 regulate the length of meiS [12], our findings indicated that the activation of the earliest replicating origins is not directly regulated by these proteins. Deletion of either Rec8 or Spo11 did not significantly alter the profile of early pre-meiotic replication in HU. Similarly, DSB sites and chromosome core sites were distributed at random with respect to replication time in meiS, indicating no direct link between replication delays and either axis assembly or DSB formation. Therefore, we suggest that meiosis- specific cohesion and the Spo11 protein are not responsible for the altered replication timing program that we observed in meiS. Similarly, full DNA replication is not required for the formation of break-competent meiotic chromosomes. The association of the axial proteins Hop1 and Red1 can be detected in HU-treated, cdc6-mn and clb5Dclb6D cells, all of which fail to duplicate their genomes, indicating that the formation of chromosome axes does not require either DNA replication or the presence of a connected sister chromatid. Association of Hop1 and Red1 occurs at cohesin- associated regions on chromosomes and is regulated by Rec8. It has been shown that mitotic and meiotic cohesin complexes load onto chromosomes independent of DNA replication [19,38,39,53], and we propose that axial proteins are similarly able to load onto unreplicated chromosomes. We have also shown that DNA replication is not required for DSB formation, as full PLoS Genetics | www.plosgenetics.org 9 May 2012 | Volume 8 | Issue 5 | e1002643