Comparing DNA replication programs reveals large timing shifts at centromeres of endocycling cells in maize roots

Abstract

39 Plant cells undergo two types of cell cycles -the mitotic cycle in which DNA replication is 40 coupled to mitosis, and the endocycle in which DNA replication occurs in the absence of cell 41 division. To investigate DNA replication programs in these two types of cell cycles, we pulse 42 labeled intact root tips of maize (Zea mays) with 5-ethynyl-2'-deoxyuridine (EdU) and used flow 43 sorting of nuclei to examine DNA replication timing (RT) during the transition from a mitotic 44 cycle to an endocycle. Here, we compare sequence-based RT profiles and found that most 45 regions of the maize genome replicate at the same time during S phase in mitotic and 46 endocycling cells, despite the need to replicate twice as much DNA in the endocycle. However, 47 regions collectively corresponding to 2% of the genome displayed significant changes in timing 48 between the two types of cell cycles. The majority of these regions are small, with a median size 49 of 135 kb, and shift to a later RT in the endocycle. However, we found larger regions that shifted 50 RT in centromeres of seven of the ten maize chromosomes. These regions covered the majority 51 of the previously defined functional centromere in each case, which are ~1-2 Mb in size in the 52 reference genome. They replicate mainly during mid S phase in mitotic cells, but primarily in 53 late S phase of the endocycle. Strikingly, the immediately adjacent pericentromere sequences are 54 primarily late replicating in both cell cycles. Analysis of CENH3 enrichment levels in nuclei of 55 different ploidies suggested that there is only a partial replacement of CENH3 nucleosomes after 56 endocycle replication is complete. The shift to later replication of centromeres and reduced 57 CENH3 enrichment after endocycle replication is consistent with the hypothesis that centromeres 58 are being inactivated as their function is no longer needed. Wear et al. 3 59 AUTHOR SUMMARY60 In traditional cell division, or mitosis, a cell's genetic material is duplicated and then split 61 between two daughter cells. In contrast, in some specialized cell types, the DNA is duplicated a 62 second time without an intervening division step, resulting in cells that carry twice as much DNA 63 -a phenomenon called an endocycle, which is common during plant development. At each step, 64 DNA replication follows an ordered program, in which highly compacted DNA is unraveled and 65 replicated in sections at different times during the synthesis (S) phase. In plants, it is unclear 66 whether traditional and endocycle programs are the same. Using root tips of maize, we found a 67 small portion of the genome whose replication in the endocycle is shifted in time, usually to later 68 in S phase. Some of these regions are scattered around the genome, and mostly coincide with 69 active genes. However, the most prominent shifts occur in centromeres. This location is 70 noteworthy because centromeres orchestrate the process of separating duplicated chromosomes 71 into daughter cells, a function that is not needed in...