Two-ended recombination at a Flp-nickase-broken replication fork.

Collision of a replication fork with a DNA nick is thought to generate a one-ended break, fostering genomic instability. Collision of the opposing converging fork with the nick could, in principle, form a second DNA end, enabling conservative repair by homologous recombination (HR). To study mechanisms of nickase-induced HR, we developed the Flp recombinase "step arrest" nickase in mammalian cells. Flp-nickase-induced HR entails two-ended, BRCA2/RAD51-dependent short tract gene conversion (STGC), BRCA2/RAD51-independent long tract gene conversion, and discoordinated two-ended invasions. HR induced by a replication-independent break and by the Flp-nickase differ in their dependence on BRCA1 . To determine the origin of the second DNA end during Flp-nickase-induced STGC, we blocked the opposing fork using a site-specific Tus/ Ter replication fork barrier. Flp-nickase-induced STGC remained robust and two-ended. Thus, collision of a single replication fork with a Flp-nick can trigger two-ended HR, possibly reflecting replicative bypass of lagging strand nicks. This response may limit genomic instability during replication of a nicked DNA template.

Novel WRN Helicase Inhibitors Selectively Target Microsatellite Unstable Cancer Cells.

Microsatellite-unstable (MSI) cancers require WRN helicase to resolve replication stress due to expanded DNA (TA)n-dinucleotide repeats. WRN is a promising synthetic lethal target for MSI tumours, and WRN inhibitors are in development. Here, we used CRISPR-Cas9 base editing to map WRN residues critical for MSI cells, validating the helicase domain as the primary drug target. Fragment-based screening led to the development of potent and highly selective WRN helicase covalent inhibitors. These compounds selectively suppressed MSI model growth In vitro and In vivo by mimicking WRN loss, inducing DNA double-strand breaks at expanded TA-repeats and DNA damage. Assessment of biomarkers in preclinical models linked TA-repeat expansions and mismatch repair (MMR) alterations to compound activity. Efficacy was confirmed in immunotherapy-resistant organoids and patient-derived xenograft (PDX) models. The discovery of potent, selective covalent WRN inhibitors provides proof of concept for synthetic-lethal targeting of WRN in MSI cancer and tools to dissect WRN biology.

Transcription as source of genetic heterogeneity in budding yeast.

Transcription presents challenges to genome stability both directly, by altering genome topology and exposing single-stranded DNA to chemical insults and nucleases, and indirectly by introducing obstacles to the DNA replication machinery. Such obstacles include the RNA polymerase holoenzyme itself, DNA-bound regulatory factors, G-quadruplexes and RNA-DNA hybrid structures known as R-loops. Here, we review the detrimental impacts of transcription on genome stability in budding yeast, as well as the mitigating effects of transcription-coupled nucleotide excision repair and of systems that maintain DNA replication fork processivity and integrity. Interactions between DNA replication and transcription have particular potential to induce mutation and structural variation, but we conclude that such interactions must have only minor effects on DNA replication by the replisome with little if any direct mutagenic outcome. However, transcription can significantly impair the fidelity of replication fork rescue mechanisms, particularly Break Induced Replication, which is used to restart collapsed replication forks when other means fail. This leads to de novo mutations, structural variation and extrachromosomal circular DNA formation that contribute to genetic heterogeneity, but only under particular conditions and in particular genetic contexts, ensuring that the bulk of the genome remains extremely stable despite the seemingly frequent interactions between transcription and DNA replication.

Checkpoint kinase interaction with DNA polymerase alpha regulates replication progression during stress.

Background: In eukaryotes, replication stress activates a checkpoint response, which facilitates genome duplication by stabilising the replisome. How the checkpoint kinases regulate the replisome remains poorly understood. The aim of this study is to identify new targets of checkpoint kinases within the replisome during replication stress. Methods: Here we use an unbiased biotin proximity-ligation approach in Saccharomyces cerevisiae to identify new interactors and substrates of the checkpoint kinase Rad53 in vivo. Results: From this screen, we identified the replication initiation factor Sld7 as a Rad53 substrate, and Pol1, the catalytic subunit of polymerase a, as a Rad53-interactor. We showed that CDK phosphorylation of Pol1 mediates its interaction with Rad53. Combined with other interactions between Rad53 and the replisome, this Rad53-Pol1 interaction is important for viability and replisome progression during replication stress. Conclusions: Together, we explain how the interactions of Rad53 with the replisome are controlled by both replication stress and the cell cycle, and why these interactions might be important for coordinating the stabilisation of both the leading and lagging strand machineries.

Dietary change without caloric restriction maintains a youthful profile in ageing yeast.

Caloric restriction increases lifespan and improves ageing health, but it is unknown whether these outcomes can be separated or achieved through less severe interventions. Here, we show that an unrestricted galactose diet in early life minimises change during replicative ageing in budding yeast, irrespective of diet later in life. Average mother cell division rate is comparable between glucose and galactose diets, and lifespan is shorter on galactose, but markers of senescence and the progressive dysregulation of gene expression observed on glucose are minimal on galactose, showing that these are not intrinsic aspects of replicative ageing but rather associated processes. Respiration on galactose is critical for minimising hallmarks of ageing, and forced respiration during ageing on glucose by overexpression of the mitochondrial biogenesis factor Hap4 also has the same effect though only in a fraction of cells. This fraction maintains Hap4 activity to advanced age with low senescence and a youthful gene expression profile, whereas other cells in the same population lose Hap4 activity, undergo dramatic dysregulation of gene expression and accumulate fragments of chromosome XII (ChrXIIr), which are tightly associated with senescence. Our findings support the existence of two separable ageing trajectories in yeast. We propose that a complete shift to the healthy ageing mode can be achieved in wild-type cells through dietary change in early life without caloric restriction.

Senescence in yeast is associated with amplified linear fragments of chromosome XII rather than ribosomal DNA circle accumulation.

The massive accumulation of extrachromosomal ribosomal DNA circles (ERCs) in yeast mother cells has been long cited as the primary driver of replicative ageing. ERCs arise through ribosomal DNA (rDNA) recombination, and a wealth of genetic data connects rDNA instability events giving rise to ERCs with shortened life span and other ageing pathologies. However, we understand little about the molecular effects of ERC accumulation. Here, we studied ageing in the presence and absence of ERCs, and unexpectedly found no evidence of gene expression differences that might indicate stress responses or metabolic feedback caused by ERCs. Neither did we observe any global change in the widespread disruption of gene expression that accompanies yeast ageing, altogether suggesting that ERCs are largely inert. Much of the differential gene expression that accompanies ageing in yeast was actually associated with markers of the senescence entry point (SEP), showing that senescence, rather than age, underlies these changes. Cells passed the SEP irrespective of ERCs, but we found the SEP to be associated with copy number amplification of a region of chromosome XII between the rDNA and the telomere (ChrXIIr) forming linear fragments up to approximately 1.8 Mb size, which arise in aged cells due to rDNA instability but through a different mechanism to ERCs. Therefore, although rDNA copy number increases dramatically with age due to ERC accumulation, our findings implicate ChrXIIr, rather than ERCs, as the primary driver of senescence during budding yeast ageing.

Escape from G1 arrest during acute MEK inhibition drives the acquisition of drug resistance.

Mutations and gene amplifications that confer drug resistance emerge frequently during chemotherapy, but their mechanism and timing are poorly understood. Here, we investigate BRAFV600E amplification events that underlie resistance to the MEK inhibitor selumetinib (AZD6244/ARRY-142886) in COLO205 cells, a well-characterized model for reproducible emergence of drug resistance, and show that BRAF amplifications acquired de novo are the primary cause of resistance. Selumetinib causes long-term G1 arrest accompanied by reduced expression of DNA replication and repair genes, but cells stochastically re-enter the cell cycle during treatment despite continued repression of pERK1/2. Most DNA replication and repair genes are re-expressed as cells enter S and G2; however, mRNAs encoding a subset of factors important for error-free replication and chromosome segregation, including TIPIN, PLK2 and PLK3, remain at low abundance. This suggests that DNA replication following escape from G1 arrest in drug is more error prone and provides a potential explanation for the DNA damage observed under long-term RAF-MEK-ERK1/2 pathway inhibition. To test the hypothesis that escape from G1 arrest in drug promotes de novo BRAF amplification, we exploited the combination of palbociclib and selumetinib. Combined treatment with selumetinib and a dose of palbociclib sufficient to reinforce G1 arrest in selumetinib-sensitive cells, but not to impair proliferation of resistant cells, delays the emergence of resistant colonies, meaning that escape from G1 arrest is critical in the formation of resistant clones. Our findings demonstrate that acquisition of MEK inhibitor resistance often occurs through de novo gene amplification and can be suppressed by impeding cell cycle entry in drug.

Stimulation of adaptive gene amplification by origin firing under replication fork constraint.

Adaptive mutations can cause drug resistance in cancers and pathogens, and increase the tolerance of agricultural pests and diseases to chemical treatment. When and how adaptive mutations form is often hard to discern, but we have shown that adaptive copy number amplification of the copper resistance gene CUP1 occurs in response to environmental copper due to CUP1 transcriptional activation. Here we dissect the mechanism by which CUP1 transcription in budding yeast stimulates copy number variation (CNV). We show that transcriptionally stimulated CNV requires TREX-2 and Mediator, such that cells lacking TREX-2 or Mediator respond normally to copper but cannot acquire increased resistance. Mediator and TREX-2 can cause replication stress by tethering transcribed loci to nuclear pores, a process known as gene gating, and transcription at the CUP1 locus causes a TREX-2-dependent accumulation of replication forks indicative of replication fork stalling. TREX-2-dependent CUP1 gene amplification occurs by a Rad52 and Rad51-mediated homologous recombination mechanism that is enhanced by histone H3K56 acetylation and repressed by Pol32 and Pif1. CUP1 amplification is also critically dependent on late-firing replication origins present in the CUP1 repeats, and mutations that remove or inactivate these origins strongly suppress the acquisition of copper resistance. We propose that replicative stress imposed by nuclear pore association causes replication bubbles from these origins to collapse soon after activation, leaving a tract of H3K56-acetylated chromatin that promotes secondary recombination events during elongation after replication fork re-start events. The capacity for inefficient replication origins to promote copy number variation renders certain genomic regions more fragile than others, and therefore more likely to undergo adaptive evolution through de novo gene amplification.

Genome-wide analysis of DNA replication and DNA double-strand breaks using TrAEL-seq.

Faithful replication of the entire genome requires replication forks to copy large contiguous tracts of DNA, and sites of persistent replication fork stalling present a major threat to genome stability. Understanding the distribution of sites at which replication forks stall, and the ensuing fork processing events, requires genome-wide methods that profile replication fork position and the formation of recombinogenic DNA ends. Here, we describe Transferase-Activated End Ligation sequencing (TrAEL-seq), a method that captures single-stranded DNA 3' ends genome-wide and with base pair resolution. TrAEL-seq labels both DNA breaks and replication forks, providing genome-wide maps of replication fork progression and fork stalling sites in yeast and mammalian cells. Replication maps are similar to those obtained by Okazaki fragment sequencing; however, TrAEL-seq is performed on asynchronous populations of wild-type cells without incorporation of labels, cell sorting, or biochemical purification of replication intermediates, rendering TrAEL-seq far simpler and more widely applicable than existing replication fork direction profiling methods. The specificity of TrAEL-seq for DNA 3' ends also allows accurate detection of double-strand break sites after the initiation of DNA end resection, which we demonstrate by genome-wide mapping of meiotic double-strand break hotspots in a dmc1Δ mutant that is competent for end resection but not strand invasion. Overall, TrAEL-seq provides a flexible and robust methodology with high sensitivity and resolution for studying DNA replication and repair, which will be of significant use in determining mechanisms of genome instability.

Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry.

A simple method for extraction of high quality RNA from cells that have been fixed, stained and sorted by flow cytometry would allow routine transcriptome analysis of highly purified cell populations and single cells. However, formaldehyde fixation impairs RNA extraction and inhibits RNA amplification. Here we show that good quality RNA can be readily extracted from stained and sorted mammalian cells if formaldehyde is replaced by glyoxal-a well-characterised fixative that is widely compatible with immunofluorescent staining methods. Although both formaldehyde and glyoxal efficiently form protein-protein crosslinks, glyoxal does not crosslink RNA to proteins nor form stable RNA adducts, ensuring that RNA remains accessible and amenable to enzymatic manipulation after glyoxal fixation. We find that RNA integrity is maintained through glyoxal fixation, permeabilisation with methanol or saponin, indirect immunofluorescent staining and flow sorting. RNA can then be extracted by standard methods and processed into RNA-seq libraries using commercial kits; mRNA abundances measured by poly(A)+ RNA-seq correlate well between freshly harvested cells and fixed, stained and sorted cells. We validate the applicability of this approach to flow cytometry by staining MCF-7 cells for the intracellular G2/M-specific antigen cyclin B1 (CCNB1), and show strong enrichment for G2/M-phase cells based on transcriptomic data. Switching to glyoxal fixation with RNA-compatible staining methods requires only minor adjustments of most existing staining and sorting protocols, and should facilitate routine transcriptomic analysis of sorted cells.

Replicative aging is associated with loss of genetic heterogeneity from extrachromosomal circular DNA in Saccharomyces cerevisiae.

Circular DNA can arise from all parts of eukaryotic chromosomes. In yeast, circular ribosomal DNA (rDNA) accumulates dramatically as cells age, however little is known about the accumulation of other chromosome-derived circles or the contribution of such circles to genetic variation in aged cells. We profiled circular DNA in Saccharomyces cerevisiae populations sampled when young and after extensive aging. Young cells possessed highly diverse circular DNA populations but 94% of the circular DNA were lost after ∼15 divisions, whereas rDNA circles underwent massive accumulation to >95% of circular DNA. Circles present in both young and old cells were characterized by replication origins including circles from unique regions of the genome and repetitive regions: rDNA and telomeric Y' regions. We further observed that circles can have flexible inheritance patterns: [HXT6/7circle] normally segregates to mother cells but in low glucose is present in up to 50% of cells, the majority of which must have inherited this circle from their mother. Interestingly, [HXT6/7circle] cells are eventually replaced by cells carrying stable chromosomal HXT6 HXT6/7 HXT7 amplifications, suggesting circular DNAs are intermediates in chromosomal amplifications. In conclusion, the heterogeneity of circular DNA offers flexibility in adaptation, but this heterogeneity is remarkably diminished with age.

The adaptive potential of circular DNA accumulation in ageing cells.

Carefully maintained and precisely inherited chromosomal DNA provides long-term genetic stability, but eukaryotic cells facing environmental challenges can benefit from the accumulation of less stable DNA species. Circular DNA molecules lacking centromeres segregate randomly or asymmetrically during cell division, following non-Mendelian inheritance patterns that result in high copy number instability and massive heterogeneity across populations. Such circular DNA species, variously known as extrachromosomal circular DNA (eccDNA), microDNA, double minutes or extrachromosomal DNA (ecDNA), are becoming recognised as a major source of the genetic variation exploited by cancer cells and pathogenic eukaryotes to acquire drug resistance. In budding yeast, circular DNA molecules derived from the ribosomal DNA (ERCs) have been long known to accumulate with age, but it is now clear that aged yeast also accumulate other high-copy protein-coding circular DNAs acquired through both random and environmentally-stimulated recombination processes. Here, we argue that accumulation of circular DNA provides a reservoir of heterogeneous genetic material that can allow rapid adaptation of aged cells to environmental insults, but avoids the negative fitness impacts on normal growth of unsolicited gene amplification in the young population.

Protocols for Northern Analysis of Exosome Substrates and Other Noncoding RNAs.

Over the past decade a plethora of noncoding RNAs (ncRNAs) have been identified, initiating an explosion in RNA research. Although RNA sequencing methods provide unsurpassed insights into ncRNA distribution and expression, detailed information on structure and processing are harder to extract from sequence data. In contrast, northern blotting methods provide uniquely detailed insights into complex RNA populations but are rarely employed outside specialist RNA research groups. Such techniques are generally considered difficult for nonspecialists, which is unfortunate as substantial technical advances in the past few decades have solved the major challenges. Here we present simple, reproducible and highly robust protocols for separating glyoxylated RNA on agarose gels and heat denatured RNA on polyacrylamide-urea gels using standard laboratory electrophoresis equipment. We also provide reliable transfer and hybridization protocols that do not require optimization for most applications. Together, these should allow any molecular biology lab to elucidate the structure and processing of ncRNAs of interest.

Transcription-induced formation of extrachromosomal DNA during yeast ageing.

Extrachromosomal circular DNA (eccDNA) facilitates adaptive evolution by allowing rapid and extensive gene copy number variation and is implicated in the pathology of cancer and ageing. Here, we demonstrate that yeast aged under environmental copper accumulate high levels of eccDNA containing the copper-resistance gene CUP1. Transcription of the tandemly repeated CUP1 gene causes CUP1 eccDNA accumulation, which occurs in the absence of phenotypic selection. We have developed a sensitive and quantitative eccDNA sequencing pipeline that reveals CUP1 eccDNA accumulation on copper exposure to be exquisitely site specific, with no other detectable changes across the eccDNA complement. eccDNA forms de novo from the CUP1 locus through processing of DNA double-strand breaks (DSBs) by Sae2, Mre11 and Mus81, and genome-wide analyses show that other protein coding eccDNA species in aged yeast share a similar biogenesis pathway. Although abundant, we find that CUP1 eccDNA does not replicate efficiently, and high-copy numbers in aged cells arise through frequent formation events combined with asymmetric DNA segregation. The transcriptional stimulation of CUP1 eccDNA formation shows that age-linked genetic change varies with transcription pattern, resulting in gene copy number profiles tailored by environment.

Tri-methylation of histone H3 lysine 4 facilitates gene expression in ageing cells.

Transcription of protein coding genes is accompanied by recruitment of COMPASS to promoter-proximal chromatin, which methylates histone H3 lysine 4 (H3K4) to form H3K4me1, H3K4me2 and H3K4me3. Here, we determine the importance of COMPASS in maintaining gene expression across lifespan in budding yeast. We find that COMPASS mutations reduce replicative lifespan and cause expression defects in almost 500 genes. Although H3K4 methylation is reported to act primarily in gene repression, particularly in yeast, repressive functions are progressively lost with age while hundreds of genes become dependent on H3K4me3 for full expression. Basal and inducible expression of these genes is also impaired in young cells lacking COMPASS components Swd1 or Spp1. Gene induction during ageing is associated with increasing promoter H3K4me3, but H3K4me3 also accumulates in non-promoter regions and the ribosomal DNA. Our results provide clear evidence that H3K4me3 is required to maintain normal expression of many genes across organismal lifespan.

Gene expression hallmarks of cellular ageing.

Ageing leads to dramatic changes in the physiology of many different tissues resulting in a spectrum of pathology. Nonetheless, many lines of evidence suggest that ageing is driven by highly conserved cell intrinsic processes, and a set of unifying hallmarks of ageing has been defined. Here, we survey reports of age-linked changes in basal gene expression across eukaryotes from yeast to human and identify six gene expression hallmarks of cellular ageing: downregulation of genes encoding mitochondrial proteins; downregulation of the protein synthesis machinery; dysregulation of immune system genes; reduced growth factor signalling; constitutive responses to stress and DNA damage; dysregulation of gene expression and mRNA processing. These encompass widely reported features of ageing such as increased senescence and inflammation, reduced electron transport chain activity and reduced ribosome synthesis, but also reveal a surprising lack of gene expression responses to known age-linked cellular stresses. We discuss how the existence of conserved transcriptomic hallmarks relates to genome-wide epigenetic differences underlying ageing clocks, and how the changing transcriptome results in proteomic alterations where data is available and to variations in cell physiology characteristic of ageing. Identification of gene expression events that occur during ageing across distant organisms should be informative as to conserved underlying mechanisms of ageing, and provide additional biomarkers to assess the effects of diet and other environmental factors on the rate of ageing.

Environmental change drives accelerated adaptation through stimulated copy number variation.

Copy number variation (CNV) is rife in eukaryotic genomes and has been implicated in many human disorders, particularly cancer, in which CNV promotes both tumorigenesis and chemotherapy resistance. CNVs are considered random mutations but often arise through replication defects; transcription can interfere with replication fork progression and stability, leading to increased mutation rates at highly transcribed loci. Here we investigate whether inducible promoters can stimulate CNV to yield reproducible, environment-specific genetic changes. We propose a general mechanism for environmentally-stimulated CNV and validate this mechanism for the emergence of copper resistance in budding yeast. By analysing a large cohort of individual cells, we directly demonstrate that CNV of the copper-resistance gene CUP1 is stimulated by environmental copper. CNV stimulation accelerates the formation of novel alleles conferring enhanced copper resistance, such that copper exposure actively drives adaptation to copper-rich environments. Furthermore, quantification of CNV in individual cells reveals remarkable allele selectivity in the rate at which specific environments stimulate CNV. We define the key mechanistic elements underlying this selectivity, demonstrating that CNV is regulated by both promoter activity and acetylation of histone H3 lysine 56 (H3K56ac) and that H3K56ac is required for CUP1 CNV and efficient copper adaptation. Stimulated CNV is not limited to high-copy CUP1 repeat arrays, as we find that H3K56ac also regulates CNV in 3 copy arrays of CUP1 or SFA1 genes. The impact of transcription on DNA damage is well understood, but our research reveals that this apparently problematic association forms a pathway by which mutations can be directed to particular loci in particular environments and furthermore that this mutagenic process can be regulated through histone acetylation. Stimulated CNV therefore represents an unanticipated and remarkably controllable pathway facilitating organismal adaptation to new environments.

RNA Binding by Histone Methyltransferases Set1 and Set2.

Histone methylation at H3K4 and H3K36 is commonly associated with genes actively transcribed by RNA polymerase II (RNAPII) and is catalyzed by Saccharomyces cerevisiae Set1 and Set2, respectively. Here we report that both methyltransferases can be UV cross-linked to RNA in vivo High-throughput sequencing of the bound RNAs revealed strong Set1 enrichment near the transcription start site, whereas Set2 was distributed along pre-mRNAs. A subset of transcripts showed notably high enrichment for Set1 or Set2 binding relative to RNAPII, suggesting functional posttranscriptional interactions. In particular, Set1 was strongly bound to the SET1 mRNA, Ty1 retrotransposons, and noncoding RNAs from the ribosomal DNA (rDNA) intergenic spacers, consistent with its previously reported silencing roles. Set1 lacking RNA recognition motif 2 (RRM2) showed reduced in vivo cross-linking to RNA and reduced chromatin occupancy. In addition, levels of H3K4 trimethylation were decreased, whereas levels of dimethylation were increased. We conclude that RNA binding by Set1 contributes to both chromatin association and methyltransferase activity.

Aging yeast gain a competitive advantage on non-optimal carbon sources.

Animals, plants and fungi undergo an aging process with remarkable physiological and molecular similarities, suggesting that aging has long been a fact of life for eukaryotes and one to which our unicellular ancestors were subject. Key biochemical pathways that impact longevity evolved prior to multicellularity, and the interactions between these pathways and the aging process therefore emerged in ancient single-celled eukaryotes. Nevertheless, we do not fully understand how aging impacts the fitness of unicellular organisms, and whether such cells gain a benefit from modulating rather than simply suppressing the aging process. We hypothesized that age-related loss of fitness in single-celled eukaryotes may be counterbalanced, partly or wholly, by a transition from a specialist to a generalist life-history strategy that enhances adaptability to other environments. We tested this hypothesis in budding yeast using competition assays and found that while young cells are more successful in glucose, highly aged cells outcompete young cells on other carbon sources such as galactose. This occurs because aged yeast divide faster than young cells in galactose, reversing the normal association between age and fitness. The impact of aging on single-celled organisms is therefore complex and may be regulated in ways that anticipate changing nutrient availability. We propose that pathways connecting nutrient availability with aging arose in unicellular eukaryotes to capitalize on age-linked diversity in growth strategy and that individual cells in higher eukaryotes may similarly diversify during aging to the detriment of the organism as a whole.