Mec1 Is Activated at the Onset of Normal S Phase by Low-dNTP Pools Impeding DNA Replication.

The Mec1 and Rad53 kinases play a central role during acute replication stress in budding yeast. They are also essential for viability in normal growth conditions, but the signal that activates the Mec1-Rad53 pathway in the absence of exogenous insults is currently unknown. Here, we show that this pathway is active at the onset of normal S phase because deoxyribonucleotide triphosphate (dNTP) levels present in G1 phase may not be sufficient to support processive DNA synthesis and impede DNA replication. This activation can be suppressed experimentally by increasing dNTP levels in G1 phase. Moreover, we show that unchallenged cells entering S phase in the absence of Rad53 undergo irreversible fork collapse and mitotic catastrophe. Together, these data indicate that cells use suboptimal dNTP pools to detect the onset of DNA replication and activate the Mec1-Rad53 pathway, which in turn maintains functional forks and triggers dNTP synthesis, allowing the completion of DNA replication.

A sterol-PI(4)P exchanger modulates the Tel1/ATM axis of the DNA damage response.

Upon DNA damage, cells activate the DNA damage response (DDR) to coordinate proliferation and DNA repair. Dietary, metabolic, and environmental inputs are emerging as modulators of how DNA surveillance and repair take place. Lipids hold potential to convey these cues, although little is known about how. We observed that lipid droplet (LD) number specifically increased in response to DNA breaks. Using Saccharomyces cerevisiae and cultured human cells, we show that the selective storage of sterols into these LD concomitantly stabilizes phosphatidylinositol-4-phosphate (PI(4)P) at the Golgi, where it binds the DDR kinase ATM. In turn, this titration attenuates the initial nuclear ATM-driven response to DNA breaks, thus allowing processive repair. Furthermore, manipulating this loop impacts the kinetics of DNA damage signaling and repair in a predictable manner. Thus, our findings have major implications for tackling genetic instability pathologies through dietary and pharmacological interventions.

Mrc1 and Rad9 cooperate to regulate initiation and elongation of DNA replication in response to DNA damage.

The S-phase checkpoint maintains the integrity of the genome in response to DNA replication stress. In budding yeast, this pathway is initiated by Mec1 and is amplified through the activation of Rad53 by two checkpoint mediators: Mrc1 promotes Rad53 activation at stalled forks, and Rad9 is a general mediator of the DNA damage response. Here, we have investigated the interplay between Mrc1 and Rad9 in response to DNA damage and found that they control DNA replication through two distinct but complementary mechanisms. Mrc1 rapidly activates Rad53 at stalled forks and represses late-firing origins but is unable to maintain this repression over time. Rad9 takes over Mrc1 to maintain a continuous checkpoint signaling. Importantly, the Rad9-mediated activation of Rad53 slows down fork progression, supporting the view that the S-phase checkpoint controls both the initiation and the elongation of DNA replication in response to DNA damage. Together, these data indicate that Mrc1 and Rad9 play distinct functions that are important to ensure an optimal completion of S phase under replication stress conditions.

Homologous recombination and Mus81 promote replication completion in response to replication fork blockage.

Impediments to DNA replication threaten genome stability. The homologous recombination (HR) pathway has been involved in the restart of blocked replication forks. Here, we used a method to increase yeast cell permeability in order to study at the molecular level the fate of replication forks blocked by DNA topoisomerase I poisoning by camptothecin (CPT). Our results indicate that Rad52 and Rad51 HR factors are required to complete DNA replication in response to CPT. Recombination events occurring during S phase do not generally lead to the restart of DNA synthesis but rather protect blocked forks until they merge with convergent forks. This fusion generates structures requiring their resolution by the Mus81 endonuclease in G2 /M. At the global genome level, the multiplicity of replication origins in eukaryotic genomes and the fork protection mechanism provided by HR appear therefore to be essential to complete DNA replication in response to fork blockage.

A new role for Rrm3 in repair of replication-born DNA breakage by sister chromatid recombination.

Replication forks stall at different DNA obstacles such as those originated by transcription. Fork stalling can lead to DNA double-strand breaks (DSBs) that will be preferentially repaired by homologous recombination when the sister chromatid is available. The Rrm3 helicase is a replisome component that promotes replication upon fork stalling, accumulates at highly transcribed regions and prevents not only transcription-induced replication fork stalling but also transcription-associated hyper-recombination. This led us to explore the possible role of Rrm3 in the repair of DSBs when originating at the passage of the replication fork. Using a mini-HO system that induces mainly single-stranded DNA breaks, we show that rrm3Δ cells are defective in DSB repair. The defect is clearly seen in sister chromatid recombination, the major repair pathway of replication-born DSBs. Our results indicate that Rrm3 recruitment to replication-born DSBs is crucial for viability, uncovering a new role for Rrm3 in the repair of broken replication forks.

DDR Inc., one business, two associates.

Eukaryotic cells activate cell cycle checkpoints in response to DNA damage. In Saccharomyces cerevisiae, the DNA damage response is achieved by the activation of the sensor kinases Mec1 and Tel1 and transmitted to the effector kinase Rad53. Rad9 and Mrc1 are thought to differentially mediate the activation of Rad53 depending on the cell cycle phase. Rad9 can respond to DNA lesions throughout the cell cycle, whereas Mrc1 responds to replication impediments in S phase. It was not clear if Rad9 and Mrc1 were triggering the same response to DNA damage occurring in S phase. By carefully studying the kinetics of activation of Rad53 by different types of replication stresses, we recently showed that Rad9 and Mrc1 cooperate in time and space to trigger a unique response to DNA damage in S phase. This primarily includes the control of both DNA replication initiation and elongation. After showing that Rad9 plays a preponderant role during S phase, the data presented here provocatively suggest that Mrc1 could also mediate the activation of Rad53 outside of S phase.

Signaling pathways of replication stress in yeast.

Eukaryotic cells activate the S-phase checkpoint in response to a variety of events affecting the progression of replication forks, collectively referred to as replication stress. This signaling pathway is divided in two branches: the DNA damage checkpoint (DDC) and the DNA replication checkpoint (DRC). Both pathways are activated by the sensor kinase Mec1 and converge on the effector kinase Rad53. However, the DDC operates throughout the cell cycle and depends on the checkpoint mediator Rad9 to activate Rad53, whereas the DRC is specific to S phase and is mediated by Mrc1 and other fork components to signal replication impediments. In this review, we summarize current knowledge on these two pathways, with a focus on the budding yeast Saccharomyces cerevisiae, in which many important aspects of the replication stress response were discovered. We also discuss the differences and similarities between DDC and DRC and speculate on how these pathways cooperate to ensure the complete and faithful duplication of the yeast genome under various replication stress conditions.

The Global Burden of Esophageal Cancer: A Disability-Adjusted Life-Year Approach.

INTRODUCTION: Esophageal cancer is the eighth most common cancer worldwide and the sixth leading cause of cancer-related deaths. As a significant cause of morbidity and mortality, its burden on society has yet to be fully characterized. The aim of this study is to examine its global burden through estimation of the disability-adjusted life years (DALYs) attributable to it. METHODS: Global incidence and mortality estimates for esophageal cancer were obtained from the International Agency for Research on Cancer GLOBOCAN 2008 database. DALYs were calculated, using methodology established by the World Health Organization. RESULTS: In 2008, 3,955,919 DALYs were attributed to esophageal cancer, at a global rate of 0.58 DALYs per 1000 people annually. Years of life lost (YLL) accounted for 96.8 % of DALYs, while years lived with disability (YLD) accounted for 3.2 %. 83.8 % of the global DALYs occurred in less-developed countries, with most accrued in Eastern Asia, comprising 50.9 % of the total. The highest rate of DALY accrual was in Southern Africa, at 1.62 DALYs per 1000 people annually. CONCLUSIONS: A substantial number of years of life were lost or affected by esophageal cancer worldwide in 2008, with the burden resting disproportionately on less-developed countries. Geographically, the greatest burden is in Eastern Asia. The vast majority of DALYs were due to YLL, rather than YLD, indicating the need to focus resources on disease prevention and early detection. Our findings provide an additional basis upon which to formulate global priorities for interventions that affect DALY reduction in esophageal cancer.

Yeast pol4 promotes tel1-regulated chromosomal translocations.

DNA double-strand breaks (DSBs) are one of the most dangerous DNA lesions, since their erroneous repair by nonhomologous end-joining (NHEJ) can generate harmful chromosomal rearrangements. PolX DNA polymerases are well suited to extend DSB ends that cannot be directly ligated due to their particular ability to bind to and insert nucleotides at the imperfect template-primer structures formed during NHEJ. Herein, we have devised genetic assays in yeast to induce simultaneous DSBs in different chromosomes in vivo. The repair of these breaks in trans could result in reciprocal chromosomal translocations that were dependent on classical Ku-dependent NHEJ. End-joining events leading to translocations were mainly based on the formation of short base pairing between 3'-overhanging DNA ends coupled to gap-filling DNA synthesis. A major proportion of these events were specifically dependent on yeast DNA polymerase Pol4 activity. In addition, we have discovered that Pol4-Thr(540) amino acid residue can be phosphorylated by Tel1/ATM kinase, which could modulate Pol4 activity during NHEJ. Our data suggest that the role of Tel1 in preventing break-induced chromosomal translocations can, to some extent, be due to its stimulating effect on gap-filling activity of Pol4 to repair DSBs in cis. Overall, this work provides further insight to the molecular mechanisms of DSB repair by NHEJ and presents a new perspective to the understanding of how chromosomal translocations are formed in eukaryotic cells.

Complex chromosomal rearrangements mediated by break-induced replication involve structure-selective endonucleases.

DNA double-strand break (DSB) repair occurring in repeated DNA sequences often leads to the generation of chromosomal rearrangements. Homologous recombination normally ensures a faithful repair of DSBs through a mechanism that transfers the genetic information of an intact donor template to the broken molecule. When only one DSB end shares homology to the donor template, conventional gene conversion fails to occur and repair can be channeled to a recombination-dependent replication pathway termed break-induced replication (BIR), which is prone to produce chromosome non-reciprocal translocations (NRTs), a classical feature of numerous human cancers. Using a newly designed substrate for the analysis of DSB-induced chromosomal translocations, we show that Mus81 and Yen1 structure-selective endonucleases (SSEs) promote BIR, thus causing NRTs. We propose that Mus81 and Yen1 are recruited at the strand invasion intermediate to allow the establishment of a replication fork, which is required to complete BIR. Replication template switching during BIR, a feature of this pathway, engenders complex chromosomal rearrangements when using repeated DNA sequences dispersed over the genome. We demonstrate here that Mus81 and Yen1, together with Slx4, also promote template switching during BIR. Altogether, our study provides evidence for a role of SSEs at multiple steps during BIR, thus participating in the destabilization of the genome by generating complex chromosomal rearrangements.

Top1p targeting by Fob1p at the ribosomal Replication Fork Barrier does not account for camptothecin sensitivity in Saccharomyces cerevisiae cells.

Camptothecin (CPT) is a specific inhibitor of the DNA topoisomerase I (Top1p), currently used in cancer therapy, which induces DNA damage and cell death. Top1p is highly active at the repeated ribosomal DNA locus (rDNA) to relax DNA supercoiling caused by elevated transcription and replication occurring in opposite directions. Fob1p interacts with, and stabilizes, Top1p at the rDNA Replication Fork Barrier (rRFB), where replication and transcription converge. Here, we have investigated if the absence of Fob1p and the consequent loss of Top1p specific targeting to the rRFB impact the sensitivity and the cell cycle progression of wild-type cells to CPT. We have also investigated the consequences of the absence of Fob1p in rad52∆ mutants, which are affected in the repair of CPT-induced DNA damage by homologous recombination. The results show that CPT sensitivity and the global cell cycle progression in cells exposed to CPT is not changed in the absence of Fob1p. Moreover, we have observed in fob1∆ cells treated with CPT that the homologous recombination factor Rad52p still congregates in the shape of foci in the nucleolus, which hosts the rDNA. This suggests that, in the absence of Fob1p, Top1p is still recruited to the rDNA, presumably at sequences other than the rRFB, and its inhibition by CPT leads to recombination events.

Unscheduled DNA replication in G1 causes genome instability and damage signatures indicative of replication collisions.

DNA replicates once per cell cycle. Interfering with the regulation of DNA replication initiation generates genome instability through over-replication and has been linked to early stages of cancer development. Here, we engineer genetic systems in budding yeast to induce unscheduled replication in a G1-like cell cycle state. Unscheduled G1 replication initiates at canonical S-phase origins. We quantifiy the composition of replisomes in G1- and S-phase and identified firing factors, polymerase α, and histone supply as factors that limit replication outside S-phase. G1 replication per se does not trigger cellular checkpoints. Subsequent replication during S-phase, however, results in over-replication and leads to chromosome breaks and chromosome-wide, strand-biased occurrence of RPA-bound single-stranded DNA, indicating head-to-tail replication collisions as a key mechanism generating genome instability upon G1 replication. Low-level, sporadic induction of G1 replication induces an identical response, indicating findings from synthetic systems are applicable to naturally occurring scenarios of unscheduled replication initiation.

Ethanol exposure increases mutation rate through error-prone polymerases.

Ethanol is a ubiquitous environmental stressor that is toxic to all lifeforms. Here, we use the model eukaryote Saccharomyces cerevisiae to show that exposure to sublethal ethanol concentrations causes DNA replication stress and an increased mutation rate. Specifically, we find that ethanol slows down replication and affects localization of Mrc1, a conserved protein that helps stabilize the replisome. In addition, ethanol exposure also results in the recruitment of error-prone DNA polymerases to the replication fork. Interestingly, preventing this recruitment through mutagenesis of the PCNA/Pol30 polymerase clamp or deleting specific error-prone polymerases abolishes the mutagenic effect of ethanol. Taken together, this suggests that the mutagenic effect depends on a complex mechanism, where dysfunctional replication forks lead to recruitment of error-prone polymerases. Apart from providing a general mechanistic framework for the mutagenic effect of ethanol, our findings may also provide a route to better understand and prevent ethanol-associated carcinogenesis in higher eukaryotes.

qDSB-Seq is a general method for genome-wide quantification of DNA double-strand breaks using sequencing.

DNA double-strand breaks (DSBs) are among the most lethal types of DNA damage and frequently cause genome instability. Sequencing-based methods for mapping DSBs have been developed but they allow measurement only of relative frequencies of DSBs between loci, which limits our understanding of the physiological relevance of detected DSBs. Here we propose quantitative DSB sequencing (qDSB-Seq), a method providing both DSB frequencies per cell and their precise genomic coordinates. We induce spike-in DSBs by a site-specific endonuclease and use them to quantify detected DSBs (labeled, e.g., using i-BLESS). Utilizing qDSB-Seq, we determine numbers of DSBs induced by a radiomimetic drug and replication stress, and reveal two orders of magnitude differences in DSB frequencies. We also measure absolute frequencies of Top1-dependent DSBs at natural replication fork barriers. qDSB-Seq is compatible with various DSB labeling methods in different organisms and allows accurate comparisons of absolute DSB frequencies across samples.

i-BLESS is an ultra-sensitive method for detection of DNA double-strand breaks.

Maintenance of genome stability is a key issue for cell fate that could be compromised by chromosome deletions and translocations caused by DNA double-strand breaks (DSBs). Thus development of precise and sensitive tools for DSBs labeling is of great importance for understanding mechanisms of DSB formation, their sensing and repair. Until now there has been no high resolution and specific DSB detection technique that would be applicable to any cells regardless of their size. Here, we present i-BLESS, a universal method for direct genome-wide DNA double-strand break labeling in cells immobilized in agarose beads. i-BLESS has three key advantages: it is the only unbiased method applicable to yeast, achieves a sensitivity of one break at a given position in 100,000 cells, and eliminates background noise while still allowing for fixation of samples. The method allows detection of ultra-rare breaks such as those forming spontaneously at G-quadruplexes.

Mismatch tolerance by DNA polymerase Pol4 in the course of nonhomologous end joining in Saccharomyces cerevisiae.

In yeast, the nonhomologous end joining pathway (NHEJ) mobilizes the DNA polymerase Pol4 to repair DNA double-strand breaks when gap filling is required prior to ligation. Using telomere-telomere fusions caused by loss of the telomeric protein Rap1 and double-strand break repair on transformed DNA as assays for NHEJ between fully uncohesive ends, we show that Pol4 is able to extend a 3'-end whose last bases are mismatched, i.e., mispaired or unpaired, to the template strand.

Multiple pathways inhibit NHEJ at telomeres.

The nonhomologous end-joining (NHEJ) repair pathway is inhibited at telomeres, preventing chromosome fusion. In budding yeast Saccharomyces cerevisiae, the Rap1 protein directly binds the telomere sequences and is required for NHEJ inhibition. Here we show that the Rap1 C-terminal domain establishes two parallel inhibitory pathways through the proteins Rif2 and Sir4. In addition, the central domain of Rap1 inhibits NHEJ independently of Rif2 and Sir4. Thus, Rap1 establishes several independent pathways to prevent telomere fusions. We discuss a possible mechanism that would explain Rif2 multifunctionality at telomeres and the recent evolutionary origin of Rif2 from an origin recognition complex (ORC) subunit.

Rap1 prevents telomere fusions by nonhomologous end joining.

Telomeres protect chromosomes from end-to-end fusions. In yeast Saccharomyces cerevisiae, the protein Rap1 directly binds telomeric DNA. Here, we use a new conditional allele of RAP1 and show that Rap1 loss results in frequent fusions between telomeres. Analysis of the fusion point with restriction enzymes indicates that fusions occur between telomeres of near wild-type length. Telomere fusions are not observed in cells lacking factors required for nonhomologous end joining (NHEJ), including Lig4 (ligase IV), KU and the Mre11 complex. SAE2 and TEL1 do not affect the frequency of fusions. Together, these results show that Rap1 is essential to block NHEJ between telomeres. Since the presence of Rap1 at telomeres has been conserved through evolution, the establishment of NHEJ suppression by Rap1 could be universal.