Hydroxyurea (HU) treatment activates the intra-S phase checkpoint proteins Cds1 and Mrc1 to prevent replication fork collapse. We found that prolonged DNA synthesis occurs in cds1⌬ and mrc1⌬ checkpoint mutants in the presence of HU and continues after release. This is coincident with increased DNA damage measured by phosphorylated histone H2A in whole cells during release. High-resolution live-cell imaging shows that mutants first accumulate extensive replication protein A (RPA) foci, followed by increased Rad52. Both DNA synthesis and RPA accumulation require the MCM helicase. We propose that a replication fork "collapse point" in HU-treated cells describes the point at which accumulated DNA damage and instability at individual forks prevent further replication. After this point, cds1⌬ and mrc1⌬ forks cannot complete genome replication. These observations establish replication fork collapse as a dynamic process that continues after release from HU block. Fission yeast Schizosaccharomyces pombe cells treated with hydroxyurea (HU) suffer nucleotide depletion and activate the intra-S phase checkpoint. This causes reversible cell cycle arrest in early S phase and prevents firing of late replication origins (12,19,62). The intra-S phase checkpoint stabilizes existing DNA replication forks and promotes recovery and replication resumption upon release. Loss of the intra-S phase checkpoint corrupts replication forks, leading to DNA damage and cell death. However, the events leading to successful recovery or fork collapse remain poorly defined.HU-treated replication forks synthesize 5 to 10 kb of DNA before they slow in budding yeast (4,27,49). Evidence from several model systems suggests that MCM helicase and polymerases are briefly uncoupled during fork slowing, which leads to increased DNA unwinding and accumulation of replication protein A (RPA) on single-stranded DNA (ssDNA) (37,39,42,43). This promotes Rad3 (ATR, or ScMec1) kinase activation (36, 66), which activates Cds1 kinase (ScRad53) (26). Cds1 inhibits the origin-activating Hsk1 kinase (ScCdc7) to block late origin firing (24,51,54), and the Mcm4 subunit of the MCM helicase is a likely Cds1 substrate (5). Cds1 also phosphorylates the Mus81 nuclease, preventing accumulation of toxic structures (7,22). Finally, Cds1 phosphorylates Cdc25, preventing mitosis in the presence of damage (6,26,65).The mediator protein Mrc1 recruits Cds1 to Rad3 to promote efficient checkpoint activation, but Mrc1 is also a nonessential component of the replication fork complex (56,57,63,64). Mrc1 couples the leading-strand DNA polymerase epsilon (Polε) to the MCM helicase (23,28,41) and regulates recombination during replication stalling and restart (1,22,47).The absence of either Cds1/ScRad53 or Rad3/ScMec1 during HU treatment leads to late origin firing, DNA unwinding, and cell death (2,12,26,49,52). This is accompanied by DNA doublestrand breaks (DSBs), histone H2A phosphorylation (phospho-H2A; H2Ax in metazoans), and accumulation of the Rad52 recombination protein in repair foci...

Unable to complete S phase, a fission yeast MCM mutant evades the mitotic checkpoint, causing aneuploidy, chromosome fragments, and bridges. The formation of apparent yeast micronuclei that are membrane bound is shown in real time; they develop DNA damage signals and may rejoin the parent nucleus.

Summary The centromere of many eukaryotes contains highly repetitive sequences marked by methylation of histone H3K9 by Clr4KMT1. This recruits multiple heterochromatin proteins including Swi6 and Chp1 to form a rigid centromere and ensure accurate chromosome segregation. In the absence of heterochromatin, cells show increased rate of recombination in the centromere, and chromosome loss. These defects are severely aggravated by loss of replication fork stability. Thus, heterochromatin proteins and replication fork protection mechanisms work in concert to prevent abnormal recombination, preserve centromere integrity, and ensure faithful chromosome segregation.

Nucleoside analogs are frequently used to label newly synthesized DNA. These analogs are toxic in many cells, with the exception of the budding yeast. We show that Schizosaccharomyces pombe behaves similarly to metazoans in response to analogs 5-bromo-29-deoxyuridine (BrdU) and 5-ethynyl-29-deoxyuridine (EdU). Incorporation causes DNA damage that activates the damage checkpoint kinase Chk1 and sensitizes cells to UV light and other DNA-damaging drugs. Replication checkpoint mutant cds1Δ shows increased DNA damage response after exposure. Finally, we demonstrate that the response to BrdU is influenced by the ribonucleotide reductase inhibitor, Spd1, suggesting that BrdU causes dNTP pool imbalance in fission yeast, as in metazoans. Consistent with this, we show that excess thymidine induces G1 arrest in wild-type fission yeast expressing thymidine kinase. Thus, fission yeast responds to nucleoside analogs similarly to mammalian cells, which has implications for their use in replication and damage research, as well as for dNTP metabolism.

Schizosaccharomyces pombe shares many genes and proteins with humans and is a good model for chromosome behavior and DNA dynamics, which can be analyzed by visualizing the behavior of fluorescently tagged proteins in vivo. Performing a genome-wide screen for changes in such proteins requires developing methods that automate analysis of a large amount of images, the first step of which requires robust segmentation of the cell. We developed a segmentation system, PombeX, that can segment cells from transmitted illumination images with focus gradient and varying contrast. Corrections for focus gradient are applied to the image to aid in accurate detection of cell membrane and cytoplasm pixels, which is used to generate initial contours for cells. Gradient vector flow snake evolution is used to obtain the final cell contours. Finally, a machine learning-based validation of cell contours removes most incorrect or spurious contours. Quantitative evaluations show overall good segmentation performance on a large set of images, regardless of differences in image quality, lighting condition, focus condition and phenotypic profile. Comparisons with recent related methods for yeast cells show that PombeX outperforms current methods, both in terms of segmentation accuracy and computational speed.