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

Plant cells undergo two types of cell cycles-the mitotic cycle in which DNA replication is coupled to mitosis, and the endocycle in which DNA replication occurs in the absence of cell division. To investigate DNA replication programs in these two types of cell cycles, we pulse labeled intact root tips of maize (Zea mays) with 5-ethynyl-2'-deoxyuridine (EdU) and used flow sorting of nuclei to examine DNA replication timing (RT) during the transition from a mitotic cycle to an endocycle. Comparison of the sequence-based RT profiles showed that most regions of the maize genome replicate at the same time during S phase in mitotic and endocycling cells, despite the need to replicate twice as much DNA in the endocycle and the fact that endocycling is typically associated with cell differentiation. However, regions collectively corresponding to 2% of the genome displayed significant changes in timing between the two types of cell cycles. The majority of these regions are small with a median size of 135 kb, shift to a later RT in the endocycle, and are enriched for genes expressed in the root tip. We found larger regions that shifted RT in centromeres of seven of the ten maize chromosomes. These regions covered the majority of the previously defined functional centromere, which ranged between 1 and 2 Mb in size in the reference genome. They replicate mainly during mid S phase in mitotic cells but primarily in late S phase of the endocycle. In contrast, the immediately adjacent pericentromere sequences are primarily late replicating in both cell cycles. Analysis of CENH3 enrichment levels in 8C vs 2C nuclei suggested that there is only a partial replacement of CENH3 nucleosomes after endocycle replication is complete. The shift to later replication of centromeres and possible reduction in CENH3 enrichment after endocycle replication is consistent with a hypothesis that centromeres are inactivated when their function is no longer needed. Author summary In traditional cell division, or mitosis, a cell's genetic material is duplicated and then split between two daughter cells. In contrast, in some specialized cell types, the DNA is duplicated a second time without an intervening division step, resulting in cells that carry twice as much DNA. This phenomenon, which is called the endocycle, is common during plant development. At each step, DNA replication follows an ordered program in which highly compacted DNA is unraveled and replicated in sections at different times during the synthesis (S) phase. In plants, it is unclear whether traditional and endocycle programs are the same, especially since endocycling cells are typically in the process of differentiation. Using root tips of maize, we found that in comparison to replication in the mitotic cell cycle, there is a small portion of the genome whose replication in the endocycle is shifted in time, usually to later in S phase. Some of these regions are scattered around the genome and mostly coincide with active genes. However, the most prominent shifts occur in centromeres. The shift to later replication in centromeres is noteworthy because they orchestrate the process of separating duplicated chromosomes into daughter cells, a function that is not needed in the endocycle.