Homologous recombination suppresses transgenerational DNA end resection and chromosomal instability in fission yeast.

Chromosomal instability (CIN) drives cell-to-cell heterogeneity, and the development of genetic diseases, including cancer. Impaired homologous recombination (HR) has been implicated as a major driver of CIN, however, the underlying mechanism remains unclear. Using a fission yeast model system, we establish a common role for HR genes in suppressing DNA double-strand break (DSB)-induced CIN. Further, we show that an unrepaired single-ended DSB arising from failed HR repair or telomere loss is a potent driver of widespread CIN. Inherited chromosomes carrying a single-ended DSB are subject to cycles of DNA replication and extensive end-processing across successive cell divisions. These cycles are enabled by Cullin 3-mediated Chk1 loss and checkpoint adaptation. Subsequent propagation of unstable chromosomes carrying a single-ended DSB continues until transgenerational end-resection leads to fold-back inversion of single-stranded centromeric repeats and to stable chromosomal rearrangements, typically isochromosomes, or to chromosomal loss. These findings reveal a mechanism by which HR genes suppress CIN and how DNA breaks that persist through mitotic divisions propagate cell-to-cell heterogeneity in the resultant progeny.

Using canavanine resistance to measure mutation rates in Schizosaccharomyces pombe.

We constructed a panel of S. pombe strains expressing DNA polymerase ε variants associated with cancer, specifically POLES297F, POLEV411L, POLEL424V, POLES459F, and used these to compare mutation rates determined by canavanine resistance with other selective methods. Canavanine-resistance mutation rates are broadly similar to those seen with reversion of the ade-485 mutation to adenine prototrophy, but lower than 5-fluoroorotic acid (FOA)-resistance rates (inactivation of ura4+ or ura5+ genes). Inactivation of several genes has been associated with canavanine resistance in S. pombe but surprisingly whole genome sequencing showed that 8/8 spontaneous canavanine-resistant mutants have an R175C mutation in the any1/arn1 gene. This gene encodes an α-arrestin-like protein involved in mediating Pub1 ubiquitylation of target proteins, and the phenotypic resistance to canavanine by this single mutation is similar to that shown by the original "can1-1" strain, which also has the any1R175C mutation. Some of the spontaneous mutants have additional mutations in arginine transporters, suggesting that this may marginally increase resistance to canavanine. The any1R175C strain showed internalisation of the Cat1 arginine transporter as previously reported, explaining the canavanine-resistance phenotype.

Expression of the cancer-associated DNA polymerase ε P286R in fission yeast leads to translesion synthesis polymerase dependent hypermutation and defective DNA replication.

Somatic and germline mutations in the proofreading domain of the replicative DNA polymerase ε (POLE-exonuclease domain mutations, POLE-EDMs) are frequently found in colorectal and endometrial cancers and, occasionally, in other tumours. POLE-associated cancers typically display hypermutation, and a unique mutational signature, with a predominance of C > A transversions in the context TCT and C > T transitions in the context TCG. To understand better the contribution of hypermutagenesis to tumour development, we have modelled the most recurrent POLE-EDM (POLE-P286R) in Schizosaccharomyces pombe. Whole-genome sequencing analysis revealed that the corresponding pol2-P287R allele also has a strong mutator effect in vivo, with a high frequency of base substitutions and relatively few indel mutations. The mutations are equally distributed across different genomic regions, but in the immediate vicinity there is an asymmetry in AT frequency. The most abundant base-pair changes are TCT > TAT transversions and, in contrast to human mutations, TCG > TTG transitions are not elevated, likely due to the absence of cytosine methylation in fission yeast. The pol2-P287R variant has an increased sensitivity to elevated dNTP levels and DNA damaging agents, and shows reduced viability on depletion of the Pfh1 helicase. In addition, S phase is aberrant and RPA foci are elevated, suggestive of ssDNA or DNA damage, and the pol2-P287R mutation is synthetically lethal with rad3 inactivation, indicative of checkpoint activation. Significantly, deletion of genes encoding some translesion synthesis polymerases, most notably Pol κ, partially suppresses pol2-P287R hypermutation, indicating that polymerase switching contributes to this phenotype.

An essential role for dNTP homeostasis following CDK-induced replication stress.

Replication stress is a common feature of cancer cells, and thus a potentially important therapeutic target. Here, we show that cyclin-dependent kinase (CDK)-induced replication stress, resulting from Wee1 inactivation, is synthetic lethal with mutations disrupting dNTP homeostasis in fission yeast. Wee1 inactivation leads to increased dNTP demand and replication stress through CDK-induced firing of dormant replication origins. Subsequent dNTP depletion leads to inefficient DNA replication, DNA damage and to genome instability. Cells respond to this replication stress by increasing dNTP supply through histone methyltransferase Set2-dependent MBF-induced expression of Cdc22, the catalytic subunit of ribonucleotide reductase (RNR). Disrupting dNTP synthesis following Wee1 inactivation, through abrogating Set2-dependent H3K36 tri-methylation or DNA integrity checkpoint inactivation results in critically low dNTP levels, replication collapse and cell death, which can be rescued by increasing dNTP levels. These findings support a 'dNTP supply and demand' model in which maintaining dNTP homeostasis is essential to prevent replication catastrophe in response to CDK-induced replication stress.

Set2 Methyltransferase Facilitates DNA Replication and Promotes Genotoxic Stress Responses through MBF-Dependent Transcription.

Chromatin modification through histone H3 lysine 36 methylation by the SETD2 tumor suppressor plays a key role in maintaining genome stability. Here, we describe a role for Set2-dependent H3K36 methylation in facilitating DNA replication and the transcriptional responses to both replication stress and DNA damage through promoting MluI cell-cycle box (MCB) binding factor (MBF)-complex-dependent transcription in fission yeast. Set2 loss leads to reduced MBF-dependent ribonucleotide reductase (RNR) expression, reduced deoxyribonucleoside triphosphate (dNTP) synthesis, altered replication origin firing, and a checkpoint-dependent S-phase delay. Accordingly, prolonged S phase in the absence of Set2 is suppressed by increasing dNTP synthesis. Furthermore, H3K36 is di- and tri-methylated at these MBF gene promoters, and Set2 loss leads to reduced MBF binding and transcription in response to genotoxic stress. Together, these findings provide new insights into how H3K36 methylation facilitates DNA replication and promotes genotoxic stress responses in fission yeast.

A Critical Balance: dNTPs and the Maintenance of Genome Stability.

A crucial factor in maintaining genome stability is establishing deoxynucleoside triphosphate (dNTP) levels within a range that is optimal for chromosomal replication. Since DNA replication is relevant to a wide range of other chromosomal activities, these may all be directly or indirectly affected when dNTP concentrations deviate from a physiologically normal range. The importance of understanding these consequences is relevant to genetic disorders that disturb dNTP levels, and strategies that inhibit dNTP synthesis in cancer chemotherapy and for treatment of other disorders. We review here how abnormal dNTP levels affect DNA replication and discuss the consequences for genome stability.

A panoply of errors: polymerase proofreading domain mutations in cancer.

Although it has long been recognized that the exonucleolytic proofreading activity intrinsic to the replicative DNA polymerases Pol δ and Pol ε is essential for faithful replication of DNA, evidence that defective DNA polymerase proofreading contributes to human malignancy has been limited. However, recent studies have shown that germline mutations in the proofreading domains of Pol δ and Pol ε predispose to cancer, and that somatic Pol ε proofreading domain mutations occur in multiple sporadic tumours, where they underlie a phenotype of 'ultramutation' and favourable prognosis. In this Review, we summarize the current understanding of the mechanisms and consequences of polymerase proofreading domain mutations in human malignancies, and highlight the potential utility of these variants as novel cancer biomarkers and therapeutic targets.

Real-time imaging of DNA damage in yeast cells using ultra-short near-infrared pulsed laser irradiation.

Analysis of accumulation of repair and checkpoint proteins at repair sites in yeast nuclei has conventionally used chemical agents, ionizing radiation or induction of endonucleases to inflict localized damage. In addition to these methods, similar studies in mammalian cells have used laser irradiation, which has the advantage that damage is inflicted at a specific nuclear region and at a precise time, and this allows accurate kinetic analysis of protein accumulation at DNA damage sites. We show here that it is feasible to use short pulses of near-infrared laser irradiation to inflict DNA damage in subnuclear regions of yeast nuclei by multiphoton absorption. In conjunction with use of fluorescently-tagged proteins, this allows quantitative analysis of protein accumulation at damage sites within seconds of damage induction. PCNA accumulated at damage sites rapidly, such that maximum accumulation was seen approximately 50 s after damage, then levels declined linearly over 200-1000 s after irradiation. RPA accumulated with slower kinetics such that hardly any accumulation was detected within 60 s of irradiation, and levels subsequently increased linearly over the next 900 s, after which levels were approximately constant (up to ca. 2700 s) at the damage site. This approach complements existing methodologies to allow analysis of key damage sensors and chromatin modification changes occurring within seconds of damage inception.

Cellular regulation of ribonucleotide reductase in eukaryotes.

Synthesis of deoxynucleoside triphosphates (dNTPs) is essential for both DNA replication and repair and a key step in this process is catalyzed by ribonucleotide reductases (RNRs), which reduce ribonucleotides (rNDPs) to their deoxy forms. Tight regulation of RNR is crucial for maintaining the correct levels of all four dNTPs, which is important for minimizing the mutation rate and avoiding genome instability. Although allosteric control of RNR was the first discovered mechanism involved in regulation of the enzyme, other controls have emerged in recent years. These include regulation of expression of RNR genes, proteolysis of RNR subunits, control of the cellular localization of the small RNR subunit, and regulation of RNR activity by small protein inhibitors. This review will focus on these additional mechanisms of control responsible for providing a balanced supply of dNTPs.

Sequential steps in DNA replication are inhibited to ensure reduction of ploidy in meiosis.

Meiosis involves two successive rounds of chromosome segregation without an intervening S phase. Exit from meiosis I is distinct from mitotic exit, in that replication origins are not licensed by Mcm2-7 chromatin binding, but spindle disassembly occurs during a transient interphase-like state before meiosis II. The absence of licensing is assumed to explain the block to DNA replication, but this has not been formally tested. Here we attempt to subvert this block by expressing the licensing control factors Cdc18 and Cdt1 during the interval between meiotic nuclear divisions. Surprisingly, this leads only to a partial round of DNA replication, even when these factors are overexpressed and effect clear Mcm2-7 chromatin binding. Combining Cdc18 and Cdt1 expression with modulation of cyclin-dependent kinase activity, activation of Dbf4-dependent kinase, or deletion of the Spd1 inhibitor of ribonucleotide reductase has little additional effect on the extent of DNA replication. Single-molecule analysis indicates this partial round of replication results from inefficient progression of replication forks, and thus both initiation and elongation replication steps may be inhibited in late meiosis. In addition, DNA replication or damage during the meiosis I-II interval fails to arrest meiotic progress, suggesting absence of checkpoint regulation of meiosis II entry.

Germline mutations affecting the proofreading domains of POLE and POLD1 predispose to colorectal adenomas and carcinomas.

Many individuals with multiple or large colorectal adenomas or early-onset colorectal cancer (CRC) have no detectable germline mutations in the known cancer predisposition genes. Using whole-genome sequencing, supplemented by linkage and association analysis, we identified specific heterozygous POLE or POLD1 germline variants in several multiple-adenoma and/or CRC cases but in no controls. The variants associated with susceptibility, POLE p.Leu424Val and POLD1 p.Ser478Asn, have high penetrance, and POLD1 mutation was also associated with endometrial cancer predisposition. The mutations map to equivalent sites in the proofreading (exonuclease) domain of DNA polymerases ɛ and δ and are predicted to cause a defect in the correction of mispaired bases inserted during DNA replication. In agreement with this prediction, the tumors from mutation carriers were microsatellite stable but tended to acquire base substitution mutations, as confirmed by yeast functional assays. Further analysis of published data showed that the recently described group of hypermutant, microsatellite-stable CRCs is likely to be caused by somatic POLE mutations affecting the exonuclease domain.

Conditional inactivation of replication proteins in fission yeast using hormone-binding domains.

The fission yeast Schizosaccharomyces pombe is a useful model for analysing DNA replication as genetic methods to allow conditional inactivation of relevant proteins can provide important information about S-phase execution. A number of strategies are available to allow regulation of protein level or activity but there are disadvantages specific to each method and this may have limitations for particular proteins or experiments. We have investigated the utility of the inducible hormone-binding domain (HBD) system, which has been described in other organisms but little used in fission yeast, for the creation of conditional-lethal replication mutants. In this method, proteins are tagged with HBD and can be regulated with ß-estradiol. In this article, we describe the application of this method in fission yeast, specifically with regard to analysis of the function of GINS, an essential component of the eukaryotic replicative helicase, the CMG complex.

Ribonucleotide reductase activity is coupled to DNA synthesis via proliferating cell nuclear antigen.

Synthesis of deoxynucleoside triphosphates (dNTPs) is required for both DNA replication and DNA repair and is catalyzed by ribonucleotide reductases (RNR), which convert ribonucleotides to their deoxy forms [1, 2]. Maintaining the correct levels of dNTPs for DNA synthesis is important for minimizing the mutation rate [3-7], and this is achieved by tight regulation of RNR [2, 8, 9]. In fission yeast, RNR is regulated in part by a small protein inhibitor, Spd1, which is degraded in S phase and after DNA damage to allow upregulation of dNTP supply [10-12]. Spd1 degradation is mediated by the activity of the CRL4(Cdt2) ubiquitin ligase complex [5, 13, 14]. This has been reported to be dependent on modulation of Cdt2 levels, which are cell cycle regulated, peaking in S phase, and which also increase after DNA damage in a checkpoint-dependent manner [7, 13]. We show here that Cdt2 level fluctuations are not sufficient to regulate Spd1 proteolysis and that the key step in this event is the interaction of Spd1 with the polymerase processivity factor proliferating cell nuclear antigen (PCNA), complexed onto DNA. This mechanism thus provides a direct link between DNA synthesis and RNR regulation.

Direct role for proliferating cell nuclear antigen in substrate recognition by the E3 ubiquitin ligase CRL4Cdt2.

The E3 ubiquitin ligase Cullin-ring ligase 4-Cdt2 (CRL4(Cdt2)) is emerging as an important cell cycle regulator that targets numerous proteins for destruction in S phase and after DNA damage, including Cdt1, p21, and Set8. CRL4(Cdt2) substrates contain a "PIP degron," which consists of a canonical proliferating cell nuclear antigen (PCNA) interaction motif (PIP box) and an adjacent basic amino acid. Substrates use their PIP box to form a binary complex with PCNA on chromatin and the basic residue to recruit CRL4(Cdt2) for substrate ubiquitylation. Using Xenopus egg extracts, we identify an acidic residue in PCNA that is essential to support destruction of all CRL4(Cdt2) substrates. This PCNA residue, which adjoins the basic amino acid of the bound PIP degron, is dispensable for substrate binding to PCNA but essential for CRL4(Cdt2) recruitment to chromatin. Our data show that the interaction of CRL4(Cdt2) with substrates requires molecular determinants not only in the substrate degron but also on PCNA. The results illustrate a potentially general mechanism by which E3 ligases can couple ubiquitylation to the formation of protein-protein interactions.

Cdt1 proteolysis is promoted by dual PIP degrons and is modulated by PCNA ubiquitylation.

Cdt1 plays a critical role in DNA replication regulation by controlling licensing. In Metazoa, Cdt1 is regulated by CRL4(Cdt2)-mediated ubiquitylation, which is triggered by DNA binding of proliferating cell nuclear antigen (PCNA). We show here that fission yeast Cdt1 interacts with PCNA in vivo and that DNA loading of PCNA is needed for Cdt1 proteolysis after DNA damage and in S phase. Activation of this pathway by ultraviolet (UV)-induced DNA damage requires upstream involvement of nucleotide excision repair or UVDE repair enzymes. Unexpectedly, two non-canonical PCNA-interacting peptide (PIP) motifs, which both have basic residues downstream, function redundantly in Cdt1 proteolysis. Finally, we show that poly-ubiquitylation of PCNA, which occurs after DNA damage, reduces Cdt1 proteolysis. This provides a mechanism for fine-tuning the activity of the CRL4(Cdt2) pathway towards Cdt1, allowing Cdt1 proteolysis to be more efficient in S phase than after DNA damage.

Monitoring DNA replication in fission yeast by incorporation of 5-ethynyl-2'-deoxyuridine.

We report procedures to allow incorporation and detection of 5-ethynyl-2'-deoxyuridine (EdU) in fission yeast, a thymidine analogue which has some technical advantages over use of bromodeoxyuridine. Low concentrations of EdU (1 µM) are sufficient to allow detection of incorporation in cells expressing thymidine kinase and human equilibrative nucleoside transporter 1 (hENT1). However EdU is toxic and activates the rad3-dependent checkpoint, resulting in cell cycle arrest, potentially limiting its applications for procedures which require labelling over more than one cell cycle. Limited DNA synthesis, when elongation is largely blocked by hydroxyurea, can be readily detected by EdU incorporation using fluorescence microscopy. Thus EdU should be useful for detecting early stages of S phase, or DNA synthesis associated with DNA repair and recombination.

Break-induced ATR and Ddb1-Cul4(Cdt)² ubiquitin ligase-dependent nucleotide synthesis promotes homologous recombination repair in fission yeast.

Nucleotide synthesis is a universal response to DNA damage, but how this response facilitates DNA repair and cell survival is unclear. Here we establish a role for DNA damage-induced nucleotide synthesis in homologous recombination (HR) repair in fission yeast. Using a genetic screen, we found the Ddb1-Cul4(Cdt)² ubiquitin ligase complex and ribonucleotide reductase (RNR) to be required for HR repair of a DNA double-strand break (DSB). The Ddb1-Cul4(Cdt)² ubiquitin ligase complex is required for degradation of Spd1, an inhibitor of RNR in fission yeast. Accordingly, deleting spd1(+) suppressed the DNA damage sensitivity and the reduced HR efficiency associated with loss of ddb1(+) or cdt2(+). Furthermore, we demonstrate a role for nucleotide synthesis in postsynaptic gap filling of resected ssDNA ends during HR repair. Finally, we define a role for Rad3 (ATR) in nucleotide synthesis and HR through increasing Cdt2 nuclear levels in response to DNA damage. Our findings support a model in which break-induced Rad3 and Ddb1-Cul4(Cdt)² ubiquitin ligase-dependent Spd1 degradation and RNR activation promotes postsynaptic ssDNA gap filling during HR repair.

Vgl1, a multi-KH domain protein, is a novel component of the fission yeast stress granules required for cell survival under thermal stress.

Multiple KH-domain proteins, collectively known as vigilins, are evolutionarily highly conserved proteins that are present in eukaryotic organisms from yeast to metazoa. Proposed roles for vigilins include chromosome segregation, messenger RNA (mRNA) metabolism, translation and tRNA transport. As a step toward understanding its biological function, we have identified the fission yeast vigilin, designated Vgl1, and have investigated its role in cellular response to environmental stress. Unlike its counterpart in Saccharomyces cerevisiae, we found no indication that Vgl1 is required for the maintenance of cell ploidy in Schizosaccharomyces pombe. Instead, Vgl1 is required for cell survival under thermal stress, and vgl1Δ mutants lose their viability more rapidly than wild-type cells when incubated at high temperature. As for Scp160 in S. cerevisiae, Vgl1 bound polysomes accumulated at endoplasmic reticulum (ER) but in a microtubule-independent manner. Under thermal stress, Vgl1 is rapidly relocalized from the ER to cytoplasmic foci that are distinct from P-bodies but contain stress granule markers such as poly(A)-binding protein and components of the translation initiation factor eIF3. Together, these observations demonstrated in S. pombe the presence of RNA granules with similar composition as mammalian stress granules and identified Vgl1 as a novel component that required for cell survival under thermal stress.

Using the DHFR heat-inducible degron for protein inactivation in Schizosaccharomyces pombe.

Inactivating a specific protein in vivo can yield important information about its function. One strategy previously developed in Saccharomyces cerevisiae by the Varshavsky group involves fusing a degron, derived from mouse dihydrofolate reductase, to the N-terminus of the target protein, which thereby confers temperature-sensitive degradation at the restrictive temperature. We describe here the application of this technique in the fission yeast, Schizosaccharomyces pombe.

GINS inactivation phenotypes reveal two pathways for chromatin association of replicative alpha and epsilon DNA polymerases in fission yeast.

The tetrameric GINS complex, consisting of Sld5-Psf1-Psf2-Psf3, plays an essential role in the initiation and elongation steps of eukaryotic DNA replication, although its biochemical function is unclear. Here we investigate the function of GINS in fission yeast, using fusion of Psf1 and Psf2 subunits to a steroid hormone-binding domain (HBD) to make GINS function conditional on the presence of beta-estradiol. We show that inactivation of Psf1-HBD causes a tight but rapidly reversible DNA replication arrest phenotype. Inactivation of Psf2-HBD similarly blocks premeiotic DNA replication and leads to loss of nuclear localization of another GINS subunit, Psf3. Inactivation of GINS has distinct effects on the replication origin association and chromatin binding of two of the replicative DNA polymerases. Inactivation of Psf1 leads to loss of chromatin binding of DNA polymerase epsilon, and Cdc45 is similarly affected. In contrast, chromatin association of the catalytic subunit of DNA polymerase alpha is not affected by defective GINS function. We suggest that GINS functions in a pathway that involves Cdc45 and is necessary for DNA polymerase epsilon chromatin binding, but that a separate pathway sets up the chromatin association of DNA polymerase alpha.