Replication timing is a crucial aspect of genome regulation that is strongly correlated with chromatin structure, gene expression, DNA repair, and genome evolution. Replication timing is determined by the timing of replication origin firing, which involves activation of MCM helicase complexes loaded at replication origins. Nonetheless, how the timing of such origin firing is regulated remains mysterious. Here, we show that the number of MCMs loaded at origins regulates replication timing. We show for the first time in vivo that multiple MCMs are loaded at origins. Because early origins have more MCMs loaded, they are, on average, more likely to fire early in S phase. Our results provide a mechanistic explanation for the observed heterogeneity in origin firing and help to explain how defined replication timing profiles emerge from stochastic origin firing. These results establish a framework in which further mechanistic studies on replication timing, such as the strong effect of heterochromatin, can be pursued.

Highlights Genetic gain for rice grain yield for International Rice Research Institute drought breeding program was estimated. Positive trend of 0.68 %, 0.87 %, 1.9 % under irrigated control, moderate and severe drought achieved. Superiority of new rice varieties over currently grown demonstrated on farmers’ fields. International Rice Research Institute developed rice varieties can protect farmers from crop losses under drought conditions.

• Ott1 regulates the alternative splicing of Mpl-TR, a truncated isoform of c-Mpl, which modulates Thpomediated signaling.• Mpl-TR expression impairs HSC engraftment.Thrombopoietin (Thpo) signaling through the c-Mpl receptor promotes either quiescence or proliferation of hematopoietic stem cells (HSCs) in a concentration-dependent manner; however, in vivo Thpo serum levels are responsive to platelet mass rather than HSC demands, suggesting additional regulation exists. Ott1 (Rbm15), a spliceosomal component originally identified as a fusion partner in t(1;22)-associated acute megakaryocytic leukemia, is also essential for maintaining HSC quiescence under stress. Ott1 controls the alternative splicing of a dominant negative isoform, Mpl-TR, capable of inhibiting HSC engraftment and attenuating Thpo signaling. Ott1, which associates with Hdac3 and the histone methyltransferase, Setd1b, binds to both c-Mpl RNA and chromatin and regulates H4 acetylation and H3K4me3 marks. Histone deacetylase or histone methyltransferase inhibition also increases Mpl-TR levels, suggesting that Ott1 uses an underlying epigenetic mechanism to control alternative splicing of c-Mpl. Manipulation of Ott1-dependent alternative splicing may therefore provide a novel pharmacologic avenue for regulating HSC quiescence and proliferation in response to Thpo. (Blood. 2015;125(6):941-948)

The budding yeast protein, Chl1p, is required for sister-chromatid cohesion, transcriptional silencing, rDNA recombination and aging. In this work, we show that Chl1p is also required for viability when DNA replication is stressed, either due to mutations or if cells are treated with genotoxic agents like methylmethane sulfonate (MMS) and ultraviolet (UV) rays. The chl1 mutation caused synthetic growth defects with mutations in DNA replication genes. At semi-permissive temperatures, the double mutants grew poorly, were less viable and showed nuclear fragmentation. They were, however, not limited in their bulk DNA synthesis. When chl1 cells were treated with relatively low levels of MMS in S-phase, they lost viability. The S-phase DNA damage checkpoint pathway, however, remained active in these cells. Agarose gel electrophoresis of genomic DNA isolated from wild-type and chl1 cells, after recovery from MMS treatment, suggested that the wild-type was more proficient in the repair of DNA damage than the mutant. Our work suggests that Chl1p is required for genome integrity when cells suffer endogenously or exogenously induced DNA damage.

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