Genome-wide mapping of human DNA replication by optical replication mapping supports a stochastic model of eukaryotic replication.

The heterogeneous nature of eukaryotic replication kinetics and the low efficiency of individual initiation sites make mapping the location and timing of replication initiation in human cells difficult. To address this challenge, we have developed optical replication mapping (ORM), a high-throughput single-molecule approach, and used it to map early-initiation events in human cells. The single-molecule nature of our data and a total of >2,500-fold coverage of the human genome on 27 million fibers averaging ∼300 kb in length allow us to identify initiation sites and their firing probability with high confidence. We find that the distribution of human replication initiation is consistent with inefficient, stochastic activation of heterogeneously distributed potential initiation complexes enriched in accessible chromatin. These observations are consistent with stochastic models of initiation-timing regulation and suggest that stochastic regulation of replication kinetics is a fundamental feature of eukaryotic replication, conserved from yeast to humans.

Chromosome Mis-segregation Generates Cell-Cycle-Arrested Cells with Complex Karyotypes that Are Eliminated by the Immune System.

Aneuploidy, a state of karyotype imbalance, is a hallmark of cancer. Changes in chromosome copy number have been proposed to drive disease by modulating the dosage of cancer driver genes and by promoting cancer genome evolution. Given the potential of cells with abnormal karyotypes to become cancerous, do pathways that limit the prevalence of such cells exist? By investigating the immediate consequences of aneuploidy on cell physiology, we identified mechanisms that eliminate aneuploid cells. We find that chromosome mis-segregation leads to further genomic instability that ultimately causes cell-cycle arrest. We further show that cells with complex karyotypes exhibit features of senescence and produce pro-inflammatory signals that promote their clearance by the immune system. We propose that cells with abnormal karyotypes generate a signal for their own elimination that may serve as a means for cancer cell immunosurveillance.

An estradiol-inducible promoter enables fast, graduated control of gene expression in fission yeast.

The fission yeast Schizosaccharomyces pombe lacks a diverse toolkit of inducible promoters for experimental manipulation. Available inducible promoters suffer from slow induction kinetics, limited control of expression levels and/or a requirement for defined growth medium. In particular, no S. pombe inducible promoter systems exhibit a linear dose-response, which would allow expression to be tuned to specific levels. We have adapted a fast, orthogonal promoter system with a large dynamic range and a linear dose response, based on ß-estradiol-regulated function of the human oestrogen receptor, for use in S. pombe. We show that this promoter system, termed Z3 EV, turns on quickly, can reach a maximal induction of 20-fold, and exhibits a linear dose response over its entire induction range, with few off-target effects. We demonstrate the utility of this system by regulating the mitotic inhibitor Wee1 to create a strain in which cell size is regulated by ß-estradiol concentration. This promoter system will be of great utility for experimentally regulating gene expression in fission yeast. Copyright © 2017 John Wiley & Sons, Ltd.

Replication timing and its emergence from stochastic processes.

The temporal organization of DNA replication has puzzled cell biologists since before the mechanism of replication was understood. The realization that replication timing correlates with important features, such as transcription, chromatin structure and genome evolution, and is misregulated in cancer and aging has only deepened the fascination. Many ideas about replication timing have been proposed, but most have been short on mechanistic detail. However, recent work has begun to elucidate basic principles of replication timing. In particular, mathematical modeling of replication kinetics in several systems has shown that the reproducible replication timing patterns seen in population studies can be explained by stochastic origin firing at the single-cell level. This work suggests that replication timing need not be controlled by a hierarchical mechanism that imposes replication timing from a central regulator, but instead results from simple rules that affect individual origins.

Changing of the guard: how ATM hands off DNA double-strand break signaling to ATR.

In a recent issue of Molecular Cell, Shiotani and Zou (2009) elucidate the biochemical mechanism underlying sequential ATM and ATR activation at DNA double-strand breaks, demonstrating that resection transforms ATM substrates into ATR substrates.

The fission yeast Rad32 (Mre11)-Rad50-Nbs1 complex is required for the S-phase DNA damage checkpoint.

Mre11, Rad50, and Nbs1 form a conserved heterotrimeric complex that is involved in recombination and DNA damage checkpoints. Mutations in this complex disrupt the S-phase DNA damage checkpoint, the checkpoint which slows replication in response to DNA damage, and cause chromosome instability and cancer in humans. However, how these proteins function and specifically where they act in the checkpoint signaling pathway remain crucial questions. We identified fission yeast Nbs1 by using a comparative genomic approach and showed that the genes for human Nbs1 and fission yeast Nbs1 and that for their budding yeast counterpart, Xrs2, are members of an evolutionarily related but rapidly diverging gene family. Fission yeast Nbs1, Rad32 (the homolog of Mre11), and Rad50 are involved in DNA damage repair, telomere regulation, and the S-phase DNA damage checkpoint. However, they are not required for G(2) DNA damage checkpoint. Our results suggest that a complex of Rad32, Rad50, and Nbs1 acts specifically in the S-phase branch of the DNA damage checkpoint and is not involved in general DNA damage recognition or signaling.