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Replication forks terminate at TERs and telomeres. Forks that converge or encounter transcription generate topological stress. Combining genetics, genomics, and transmission electron microscopy, we find that Rrm3 hPif1 and Sen1 hSenataxin helicases assist termination at TERs; Sen1 specifically acts at telomeres. rrm3 and sen1 genetically interact and fail to terminate replication, exhibiting fragility at termination zones (TERs) and telomeres. sen1rrm3 accumulates RNA-DNA hybrids and X-shaped gapped or reversed converging forks at TERs; sen1, but not rrm3, builds up RNA polymerase II (RNPII) at TERs and telomeres. Rrm3 and Sen1 restrain Top1 and Top2 activities, preventing toxic accumulation of positive supercoil at TERs and telomeres. We suggest that Rrm3 and Sen1 coordinate the activities of Top1 and Top2 when forks encounter transcription head on or codirectionally, respectively, thus preventing the slowing down of DNA and RNA polymerases. Hence Rrm3 and Sen1 are indispensable to generate permissive topological conditions for replication termination., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

RNA:DNA hybrids are generated naturally behind the elongating RNA polymerase as a transcriptional intermediate. However, prolonged persistence of these structures challenges the integrity of the genome by creating R-loops and by interfering with DNA replication and other chromatin related processes. Precise mapping and characterization of their distribution across the genome has been a major challenge to understand the genesis of RNA:DNA hybrids and their conversion into genotoxic intermediates. Here we provide the detailed protocol for mapping RNA:DNA hybrid across the Saccharomyces cerevisiae genome., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The DNA damage response (DDR) coordinates DNA metabolism with nuclear and non-nuclear processes. The DDR kinase Rad53 CHK1/CHK2 controls histone degradation to assist DNA repair. However, Rad53 deficiency causes histone-dependent growth defects in the absence of DNA damage, pointing out unknown physiological functions of the Rad53-histone axis. Here we show that histone dosage control by Rad53 ensures metabolic homeostasis. Under physiological conditions, Rad53 regulates histone levels through inhibitory phosphorylation of the transcription factor Spt21 NPAT on Ser276. Rad53-Spt21 mutants display severe glucose dependence, caused by excess histones through two separable mechanisms: dampening of acetyl-coenzyme A-dependent carbon metabolism through histone hyper-acetylation, and Sirtuin-mediated silencing of starvation-induced subtelomeric domains. We further demonstrate that repression of subtelomere silencing by physiological Tel1 ATM and Rpd3 HDAC activities coveys tolerance to glucose restriction. Our findings identify DDR mutations, histone imbalances and aberrant subtelomeric chromatin as interconnected causes of glucose dependence, implying that DDR kinases coordinate metabolism and epigenetic changes.

Transcription challenges the integrity of replicating chromosomes by generating topological stress and conflicts with forks 1,2 . The DNA topoisomerases Top1 and Top2 and the HMGB family protein Hmo1 assist DNA replication and transcription 3-6 . Here we describe the topological architecture of genes in Saccharomyces cerevisiae during the G1 and S phases of the cell cycle. We found under-wound DNA at gene boundaries and over-wound DNA within coding regions. This arrangement does not depend on Pol II or S phase. Top2 and Hmo1 preserve negative supercoil at gene boundaries, while Top1 acts at coding regions. Transcription generates RNA-DNA hybrids within coding regions, independently of fork orientation. During S phase, Hmo1 protects under-wound DNA from Top2, while Top2 confines Pol II and Top1 at coding units, counteracting transcription leakage and aberrant hybrids at gene boundaries. Negative supercoil at gene boundaries prevents supercoil diffusion and nucleosome repositioning at coding regions. DNA looping occurs at Top2 clusters. We propose that Hmo1 locks gene boundaries in a cruciform conformation and, with Top2, modulates the architecture of genes that retain the memory of the topological arrangements even when transcription is repressed.

Dna2 is a DNA helicase-endonuclease mediating DSB resection and Okazaki fragment processing. Dna2 ablation is lethal and rescued by inactivation of Pif1, a helicase assisting Okazaki fragment maturation, Pol32, a DNA polymerase δ subunit, and Rad9, a DNA damage response (DDR) factor. Dna2 counteracts fork reversal and promotes fork restart. Here we show that Dna2 depletion generates lethal DNA structures activating the DDR. While PIF1 deletion rescues the lethality of Dna2 depletion, RAD9 ablation relieves the first cell cycle arrest causing genotoxicity after few cell divisions. Slow fork speed attenuates DDR in Dna2 deprived cells. Electron microscopy shows that Dna2-ablated cells accumulate long ssDNA flaps behind the forks through Pif1 and fork speed. We suggest that Dna2 offsets the strand displacement activity mediated by the lagging strand polymerase and Pif1, processing long ssDNA flaps to prevent DDR activation. We propose that this Dna2 function has been hijacked by Break Induced Replication in DSB processing.

Cell survival to replication stress depends on the activation of the Mec1 ATR -Rad53 checkpoint response that protects the integrity of stalled forks and controls the origin firing program. Here we found that Mad2, a member of the spindle assembly checkpoint (SAC), contributes to efficient origin firing and to cell survival in response to replication stress. We show that Rad53 and Mad2 promote S-phase cyclin expression through different mechanisms: while Rad53 influences Clb5,6 degradation, Mad2 promotes their protein synthesis. We found that Mad2 co-sediments with polysomes and modulates the association of the translation inhibitor Caf20 4E-BP with the translation machinery and the initiation factor eIF4E. This Mad2-dependent translational regulatory process does not depend on other SAC proteins. Altogether our observations indicate that Mad2 has an additional function outside of mitosis to control DNA synthesis and collaborates with the Mec1-Rad53 regulatory axis to allow cell survival in response to replication stress., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

The yeast RNA/DNA helicase Sen1, Senataxin in human, preserves the integrity of replication forks encountering transcription by removing RNA-DNA hybrids. Here we show that, in sen1 mutants, when a replication fork clashes head-on with transcription is arrested and, as a consequence, the progression of the sister fork moving in the opposite direction within the same replicon is also impaired. Therefore, sister forks remain coupled when one of the two forks is arrested by transcription, a fate different from that experienced by forks encountering Double Strand Breaks. We also show that dormant origins of replication are activated to ensure DNA synthesis in the proximity to the forks arrested by transcription. Dormant origin firing is not inhibited by the replication checkpoint, rather dormant origins are fired if they cannot be timely inactivated by passive replication. In sen1 mutants, the Mre11 and Mrc1-Ctf4 complexes protect the forks arrested by transcription from processing mediated by the Exo1 nuclease. Thus, a harmless head-on replication-transcription clash resolution requires the fine-tuning of origin firing and coordination among Sen1, Exo1, Mre11 and Mrc1-Ctf4 complexes.

Mec1 ATR mediates the DNA damage response (DDR), integrating chromosomal signals and mechanical stimuli. We show that the PP2A phosphatases, ceramide-activated enzymes, couple cell metabolism with the DDR. Using genomic screens, metabolic analysis, and genetic and pharmacological studies, we found that PP2A attenuates the DDR and that three metabolic circuits influence the DDR by modulating PP2A activity. Irc21, a putative cytochrome b5 reductase that promotes the condensation reaction generating dihydroceramides (DHCs), and Ppm1, a PP2A methyltransferase, counteract the DDR by activating PP2A; conversely, the nutrient-sensing TORC1-Tap42 axis sustains DDR activation by inhibiting PP2A. Loss-of-function mutations in IRC21, PPM1, and PP2A and hyperactive tap42 alleles rescue mec1 mutants. Ceramides synergize with rapamycin, a TORC1 inhibitor, in counteracting the DDR. Hence, PP2A integrates nutrient-sensing and metabolic pathways to attenuate the Mec1 ATR response. Our observations imply that metabolic changes affect genome integrity and may help with exploiting therapeutic options and repositioning known drugs., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Replication stress activates the Mec1(ATR) and Rad53 kinases. Rad53 phosphorylates nuclear pores to counteract gene gating, thus preventing aberrant transitions at forks approaching transcribed genes. Here, we show that Rrm3 and Pif1, DNA helicases assisting fork progression across pausing sites, are detrimental in rad53 mutants experiencing replication stress. Rrm3 and Pif1 ablations rescue cell lethality, chromosome fragmentation, replisome-fork dissociation, fork reversal, and processing in rad53 cells. Through phosphorylation, Rad53 regulates Rrm3 and Pif1; phospho-mimicking rrm3 mutants ameliorate rad53 phenotypes following replication stress without affecting replication across pausing elements under normal conditions. Hence, the Mec1-Rad53 axis protects fork stability by regulating nuclear pores and DNA helicases. We propose that following replication stress, forks stall in an asymmetric conformation by inhibiting Rrm3 and Pif1, thus impeding lagging strand extension and preventing fork reversal; conversely, under unperturbed conditions, the peculiar conformation of forks encountering pausing sites would depend on active Rrm3 and Pif1., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Template switching (TS) mediates damage bypass via a recombination-related mechanism involving PCNA polyubiquitination and polymerase δ-dependent DNA synthesis. Using two-dimensional gel electrophoresis and EM, here we characterize TS intermediates arising in Saccharomyces cerevisiae at a defined chromosome locus, identifying five major families of intermediates. Single-stranded DNA gaps of 150-200 nt, and not DNA ends, initiate TS by strand invasion. This causes reannealing of the parental strands and exposure of the nondamaged newly synthesized chromatid, which serves as a replication template for the other blocked nascent strand. Structures resembling double Holliday junctions, postulated to be central double-strand break-repair intermediates but so far visualized only in meiosis, mediate late stages of TS before being processed to hemicatenanes. Our results reveal the DNA transitions accounting for recombination-mediated DNA-damage tolerance in mitotic cells and replication under conditions of genotoxic stress.

Transcription hinders replication fork progression and stability. The ATR checkpoint and specialized DNA helicases assist DNA synthesis across transcription units to protect genome integrity. Combining genomic and genetic approaches together with the analysis of replication intermediates, we searched for factors coordinating replication with transcription. We show that the Sen1/Senataxin DNA/RNA helicase associates with forks, promoting their progression across RNA polymerase II (RNAPII)-transcribed genes. sen1 mutants accumulate aberrant DNA structures and DNA-RNA hybrids while forks clash head-on with RNAPII transcription units. These replication defects correlate with hyperrecombination and checkpoint activation in sen1 mutants. The Sen1 function at the forks is separable from its role in RNA processing. Our data, besides unmasking a key role for Senataxin in coordinating replication with transcription, provide a framework for understanding the pathological mechanisms caused by Senataxin deficiencies and leading to the severe neurodegenerative diseases ataxia with oculomotor apraxia type 2 and amyotrophic lateral sclerosis 4., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Transcription hinders replication fork progression and stability, and the Mec1/ATR checkpoint protects fork integrity. Examining checkpoint-dependent mechanisms controlling fork stability, we find that fork reversal and dormant origin firing due to checkpoint defects are rescued in checkpoint mutants lacking THO, TREX-2, or inner-basket nucleoporins. Gene gating tethers transcribed genes to the nuclear periphery and is counteracted by checkpoint kinases through phosphorylation of nucleoporins such as Mlp1. Checkpoint mutants fail to detach transcribed genes from nuclear pores, thus generating topological impediments for incoming forks. Releasing this topological complexity by introducing a double-strand break between a fork and a transcribed unit prevents fork collapse. Mlp1 mutants mimicking constitutive checkpoint-dependent phosphorylation also alleviate checkpoint defects. We propose that the checkpoint assists fork progression and stability at transcribed genes by phosphorylating key nucleoporins and counteracting gene gating, thus neutralizing the topological tension generated at nuclear pore gated genes., (Copyright © 2011 Elsevier Inc. All rights reserved.)

THO/TREX is a conserved nuclear complex that functions in mRNP biogenesis and prevents transcription-associated recombination. Whether or not it has a ubiquitous role in the genome is unknown. Chromatin immunoprecipitation (ChIP)-chip studies reveal that the Hpr1 component of THO and the Sub2 RNA-dependent ATPase have genome-wide distributions at active ORFs in yeast. In contrast to RNA polymerase II, evenly distributed from promoter to termination regions, THO and Sub2 are absent at promoters and distributed in a gradual 5' → 3' gradient. This is accompanied by a genome-wide impact of THO-Sub2 deletions on expression of highly expressed, long and high G+C-content genes. Importantly, ChIP-chips reveal an over-recruitment of Rrm3 in active genes in THO mutants that is reduced by RNaseH1 overexpression. Our work establishes a genome-wide function for THO-Sub2 in transcription elongation and mRNP biogenesis that function to prevent the accumulation of transcription-mediated replication obstacles, including R-loops.

Protein acetylation is mediated by histone acetyltransferases (HATs) and deacetylases (HDACs), which influence chromatin dynamics, protein turnover and the DNA damage response. ATM and ATR mediate DNA damage checkpoints by sensing double-strand breaks and single-strand-DNA-RFA nucleofilaments, respectively. However, it is unclear how acetylation modulates the DNA damage response. Here we show that HDAC inhibition/ablation specifically counteracts yeast Mec1 (orthologue of human ATR) activation, double-strand-break processing and single-strand-DNA-RFA nucleofilament formation. Moreover, the recombination protein Sae2 (human CtIP) is acetylated and degraded after HDAC inhibition. Two HDACs, Hda1 and Rpd3, and one HAT, Gcn5, have key roles in these processes. We also find that HDAC inhibition triggers Sae2 degradation by promoting autophagy that affects the DNA damage sensitivity of hda1 and rpd3 mutants. Rapamycin, which stimulates autophagy by inhibiting Tor, also causes Sae2 degradation. We propose that Rpd3, Hda1 and Gcn5 control chromosome stability by coordinating the ATR checkpoint and double-strand-break processing with autophagy.

Chromosome replication initiates at multiple replicons and terminates when forks converge. In E. coli, the Tus-TER complex mediates polar fork converging at the terminator region, and aberrant termination events challenge chromosome integrity and segregation. Since in eukaryotes, termination is less characterized, we used budding yeast to identify the factors assisting fork fusion at replicating chromosomes. Using genomic and mechanistic studies, we have identified and characterized 71 chromosomal termination regions (TERs). TERs contain fork pausing elements that influence fork progression and merging. The Rrm3 DNA helicase assists fork progression across TERs, counteracting the accumulation of X-shaped structures. The Top2 DNA topoisomerase associates at TERs in S phase, and G2/M facilitates fork fusion and prevents DNA breaks and genome rearrangements at TERs. We propose that in eukaryotes, replication fork barriers, Rrm3, and Top2 coordinate replication fork progression and fusion at TERs, thus counteracting abnormal genomic transitions., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Specialized topoisomerases solve the topological constraints arising when replication forks encounter transcription. We have investigated the contribution of Top2 in S phase transcription. Specifically in S phase, Top2 binds intergenic regions close to transcribed genes. The Top2-bound loci exhibit low nucleosome density and accumulate gammaH2A when Top2 is defective. These intergenic loci associate with the HMG protein Hmo1 throughout the cell cycle and are refractory to the histone variant Htz1. In top2 mutants, Hmo1 is deleterious and accumulates at pericentromeric regions in G2/M. Our data indicate that Top2 is dispensable for transcription and that Hmo1 and Top2 bind in the proximity of genes transcribed in S phase suppressing chromosome fragility at the M-G1 transition. We propose that an Hmo1-dependent epigenetic signature together with Top2 mediate an S phase architectural pathway to preserve genome integrity.

In response to replication stress, the Mec1/ATR and SUMO pathways control stalled- and damaged-fork stability. We investigated the S phase response at forks encountering a broken template (termed the terminal fork). We show that double-strand break (DSB) formation can locally trigger dormant origin firing. Irreversible fork resolution at the break does not impede progression of the other fork in the same replicon (termed the sister fork). The Mre11-Tel1/ATM response acts at terminal forks, preventing accumulation of cruciform DNA intermediates that tether sister chromatids and can undergo nucleolytic processing. We conclude that sister forks can be uncoupled during replication and that, after DSB-induced fork termination, replication is rescued by dormant origin firing or adjacent replicons. We have uncovered a Tel1/ATM- and Mre11-dependent response controlling terminal fork integrity. Our findings have implications for those genome instability syndromes that accumulate DNA breaks during S phase and for forks encountering eroding telomeres.

Recombination is important for DNA repair, but it can also contribute to genome rearrangements. RecQ helicases, including yeast Sgs1 and human BLM, safeguard genome integrity through their functions in DNA recombination. Sgs1 prevents the accumulation of Rad51-dependent sister chromatid junctions at damaged replication forks, and its functionality seems to be regulated by Ubc9- and Mms21-dependent sumoylation. We show that mutations in Smc5-6 and Esc2 also lead to an accumulation of recombinogenic structures at damaged replication forks. Because Smc5-6 is sumoylated in an Mms21-dependent manner, this finding suggests that Smc5-6 may be a crucial target of Mms21 implicated in this process. Our data reveal that Smc5-6 and Esc2 are required to tolerate DNA damage and that their functionality is critical in genotoxic conditions in the absence of Sgs1. As reported previously for Sgs1 and Smc5-6, we find that Esc2 physically interacts with Ubc9 and SUMO. This interaction is correlated with the ability of Esc2 to promote DNA damage tolerance. Collectively, these data suggest that Esc2 and Smc5-6 act in concert with Sgs1 to prevent the accumulation of recombinogenic structures at damaged replication forks, likely by integrating sumoylation activities to regulate the repair pathways in response to damaged DNA.

Replication by template switch is thought to mediate DNA damage-bypass and fillings of gaps. Gap-filling repair requires homologous recombination as well as Rad18- and Rad5-mediated proliferating cell nuclear antigen (PCNA) polyubiquitylation. However, it is unclear whether these processes are coordinated, and the physical evidence for Rad18-Rad5-dependent template switch at replication forks is still elusive. Here we show, using genetic and physical approaches, that in budding yeast (Saccharomyces cerevisiae) Rad18 is required for the formation of X-shaped sister chromatid junctions (SCJs) at damaged replication forks through a process involving PCNA polyubiquitylation and the ubiquitin-conjugating enzymes Mms2 and Ubc13. The Rad18-Mms2-mediated damage-bypass through SCJs requires the small ubiquitin-like modifier (SUMO)-conjugating enzyme Ubc9 and SUMOylated PCNA, and is coordinated with Rad51-dependent recombination events. We propose that the Rad18-Rad5-Mms2-dependent SCJs represent template switch events. Altogether, our results unmask a role for PCNA ubiquitylation and SUMOylation pathways in promoting transient damage-induced replication-coupled recombination events involving sister chromatids at replication forks.

Replication of the eukaryotic genome is a difficult task, as cells must coordinate chromosome replication with chromatin remodeling, DNA recombination, DNA repair, transcription, cell cycle progression, and sister chromatid cohesion. Yet, DNA replication is a potentially genotoxic process, particularly when replication forks encounter a bulge in the template: forks under these conditions may stall and restart or even break down leading to fork collapse. It is now clear that fork collapse stimulates chromosomal rearrangements and therefore represents a potential source of DNA damage. Hence, the comprehension of the mechanisms that preserve replication fork integrity or that promote fork collapse are extremely relevant for the understanding of the cellular processes controlling genome stability. Here we describe some experimental approaches that can be used to physically visualize the quality of replication forks in the yeast S. cerevisiae and to distinguish between stalled and collapsed forks.

The replication checkpoint coordinates the cell cycle with DNA replication and recombination, preventing genome instability and cancer. The budding yeast Rad53 checkpoint kinase stabilizes stalled forks and replisome-fork complexes, thus preventing the accumulation of ss-DNA regions and reversed forks at collapsed forks. We searched for factors involved in the processing of stalled forks in HU-treated rad53 cells. Using the neutral-neutral two-dimensional electrophoresis technique (2D gel) and psoralen crosslinking combined with electron microscopy (EM), we found that the Exo1 exonuclease is recruited to stalled forks and, in rad53 mutants, counteracts reversed fork accumulation by generating ss-DNA intermediates. Hence, Exo1-mediated fork processing resembles the action of E. coli RecJ nuclease at damaged forks. Fork stability and replication restart are influenced by both DNA polymerase-fork association and Exo1-mediated processing. We suggest that Exo1 counteracts fork reversal by resecting newly synthesized chains and resolving the sister chromatid junctions that cause regression of collapsed forks.

A single double-strand break (DSB) induced by HO endonuclease triggers both repair by homologous recombination and activation of the Mec1-dependent DNA damage checkpoint in budding yeast. Here we report that DNA damage checkpoint activation by a DSB requires the cyclin-dependent kinase CDK1 (Cdc28) in budding yeast. CDK1 is also required for DSB-induced homologous recombination at any cell cycle stage. Inhibition of homologous recombination by using an analogue-sensitive CDK1 protein results in a compensatory increase in non-homologous end joining. CDK1 is required for efficient 5' to 3' resection of DSB ends and for the recruitment of both the single-stranded DNA-binding complex, RPA, and the Rad51 recombination protein. In contrast, Mre11 protein, part of the MRX complex, accumulates at unresected DSB ends. CDK1 is not required when the DNA damage checkpoint is initiated by lesions that are processed by nucleotide excision repair. Maintenance of the DSB-induced checkpoint requires continuing CDK1 activity that ensures continuing end resection. CDK1 is also important for a later step in homologous recombination, after strand invasion and before the initiation of new DNA synthesis.

A number of genes have been identified in the fully sequenced genome of Saccharomyces cerevisiae that appear to be conserved throughout evolution and the function of which remains poorly understood. In this manuscript we describe the initial characterization of yeast BUD31 gene. cDNA sequences highly related to BUD31 have been identified in human, Xenopus laevis, and Caenorhabditis elegans. With the aim of further understanding its function, we generated a BUD31-null yeast strain and characterized its phenotype: bud31l mutant cells showed severe cytoskeletal abnormalities, with dramatic effects on actin distribution and bud formation. We also proceeded to identify interacting proteins using the tandem affinity-purification method, coupled to mass spectrometry: Bud3lp was found in complex with proteins involved in mRNA splicing. We propose that the observed phenotypes for bud31-null strain could be the result of defective splicing and indicate a first functional role for Bud3lp and its homologs.

The replication checkpoint controls the integrity of replicating chromosomes by stabilizing stalled forks, thus preventing the accumulation of abnormal replication and recombination intermediates that contribute to genome instability. Checkpoint-defective cells are susceptible to rearrangements at chromosome fragile sites when replication pauses, and certain human cancer prone diseases suffer checkpoint abnormalities. It is unclear as to how the checkpoint stabilizes stalled forks and how cells sense replication blocks. We have analysed the checkpoint contribution in controlling replisome-fork association when replication pauses. We show that in yeast wild-type cells, stalled forks exhibit stable replisome complexes and the checkpoint sensors Ddc1 and Ddc2, thus activating Rad53 checkpoint kinase. Ddc1/Ddc2 recruitment on stalled forks and Rad53 activation are influenced by the single-strand-binding protein replication factor A (RFA). rad53 forks exhibit a defective association with DNA polymerases alpha, epsilon and delta. Further, in rad53 mutants, stalled forks progressively generate abnormal structures that turn into checkpoint signals by accumulating RFA, Ddc1 and Ddc2. We suggest that, following replication blocks, checkpoint activation mediated by RFA-ssDNA filaments stabilizes stalled forks by controlling replisome-fork association, thus preventing unscheduled recruitment of recombination enzymes that could otherwise cause the pathological processing of the forks.

Very few gene conversions in mitotic cells are associated with crossovers, suggesting that these events are regulated. This may be important for the maintenance of genetic stability. We have analyzed the relationship between homologous recombination and crossing-over in haploid budding yeast and identified factors involved in the regulation of crossover outcomes. Gene conversions unaccompanied by a crossover appear 30 min before conversions accompanied by exchange, indicating that there are two different repair mechanisms in mitotic cells. Crossovers are rare (5%), but deleting the BLM/WRN homolog, SGS1, or the SRS2 helicase increases crossovers 2- to 3-fold. Overexpressing SRS2 nearly eliminates crossovers, whereas overexpression of RAD51 in srs2Delta cells almost completely eliminates the noncrossover recombination pathway. We suggest Sgs1 and its associated topoisomerase Top3 remove double Holliday junction intermediates from a crossover-producing repair pathway, thereby reducing crossovers. Srs2 promotes the noncrossover synthesis-dependent strand-annealing (SDSA) pathway, apparently by regulating Rad51 binding during strand exchange.

In Saccharomyces strains in which homologous recombination is delayed sufficiently to activate the DNA damage checkpoint, Rad53p checkpoint kinase activity appears 1 hr after DSB induction and disappears soon after completion of repair. Cells lacking Srs2p helicase fail to recover even though they apparently complete DNA repair; Rad53p kinase remains activated. srs2Delta cells also fail to adapt when DSB repair is prevented. The recovery defect of srs2Delta is suppressed in mec1Delta strains lacking the checkpoint or when DSB repair occurs before checkpoint activation. Permanent preanaphase arrest of srs2Delta cells is reversed by the addition of caffeine after cells have arrested. Thus, in addition to its roles in recombination, Srs2p appears to be needed to turn off the DNA damage checkpoint.

Checkpoint-mediated control of replicating chromosomes is essential for preventing cancer. In yeast, Rad53 kinase protects stalled replication forks from pathological rearrangements. To characterize the mechanisms controlling fork integrity, we analyzed replication intermediates formed in response to replication blocks using electron microscopy. At the forks, wild-type cells accumulate short single-stranded regions, which likely causes checkpoint activation, whereas rad53 mutants exhibit extensive single-stranded gaps and hemi-replicated intermediates, consistent with a lagging-strand synthesis defect. Further, rad53 cells accumulate Holliday junctions through fork reversal. We speculate that, in checkpoint mutants, abnormal replication intermediates begin to form because of uncoordinated replication and are further processed by unscheduled recombination pathways, causing genome instability.

Saccharomyces cells with a single unrepaired double-strand break (DSB) will adapt to checkpoint-mediated G2/M arrest and resume cell cycle progression. The decision to adapt is finely regulated by the extent of single-stranded DNA generated from a DSB. We show that cells lacking the recombination protein Tid1p are unable to adapt, but that this defect is distinct from any role in recombination. As with the adaptation-defective mutations yku70Delta and cdc5-ad, permanent arrest in tid1Delta is bypassed by the deletion of the checkpoint gene RAD9. Permanent arrest of tid1Delta cells is suppressed by the rfa1-t11 mutation in the ssDNA binding complex RPA, similar to yku70Delta, whereas the defect in cdc5-ad is not suppressed. Unlike yku70Delta, tid1Delta does not affect 5'-to-3' degradation of DSB ends. The tid1Delta defect cannot be complemented by overexpressing the homolog Rad54p, nor is it affected in rad51Delta tid1Delta, rad54Delta tid1Delta, or rad52Delta tid1Delta double mutants that prevent essentially all homologous recombination. We suggest that Tid1p participates in monitoring the extent of single-stranded DNA produced by resection of DNA ends in a fashion that is distinct from its role in recombination.

Saccharomyces cells with one unrepaired double-strand break (DSB) adapt after checkpoint-mediated G2/M arrest. Adaptation is accompanied by loss of Rad53p checkpoint kinase activity and Chk1p phosphorylation. Rad53p kinase remains elevated in yku70delta and cdc5-ad cells that fail to adapt. Permanent G2/M arrest in cells with increased single-stranded DNA is suppressed by the rfa1-t11 mutation, but this RPA mutation does not suppress permanent arrest in cdc5-ad cells. Checkpoint kinase activation and inactivation can be followed in G2-arrested cells, but there is no kinase activation in G1-arrested cells. We conclude that activation of the checkpoint kinases in response to a single DNA break is cell cycle regulated and that adaptation is an active process by which these kinases are inactivated.

In response to genotoxic agents and cell cycle blocks all eukaryotic cells activate a set of surveillance mechanims called checkpoints. A subset of these mechanisms is represented by the DNA damage checkpoint, which is triggered by DNA lesions. The activation of this signal transduction pathway leads to a delay of cell cycle progression to prevent replication and segregation of damaged DNA molecules, and to induce transcription of several DNA repair genes. The yeast Saccharomyces cerevisiae has been invaluable in genetically dissecting the DNA damage checkpoint pathway and recent findings have provided new insights into the architecture of checkpoint protein complexes, in their order of function and in the mechanisms controlling DNA replication in response to DNA damage.

The Saccharomyces cerevisiae Rad53 protein kinase is required for the execution of checkpoint arrest at multiple stages of the cell cycle. We found that Rad53 autophosphorylation activity depends on in trans phosphorylation mediated by Mec1 and does not require physical association with other proteins. Uncoupling in trans phosphorylation from autophosphorylation using a rad53 kinase-defective mutant results in a dominant-negative checkpoint defect. Activation of Rad53 in response to DNA damage in G(1) requires the Rad9, Mec3, Ddc1, Rad17 and Rad24 checkpoint factors, while this dependence is greatly reduced in S phase cells. Furthermore, during recovery from checkpoint activation, Rad53 activity decreases through a process that does not require protein synthesis. We also found that Rad53 modulates the lagging strand replication apparatus by controlling phosphorylation of the DNA polymerase alpha-primase complex in response to intra-S DNA damage.

Eukaryotic cells have evolved a network of control mechanisms, known as checkpoints, which coordinate cell-cycle progression in response to internal and external cues. The yeast Saccharomyces cerevisiae has been invaluable in dissecting genetically the DNA damage checkpoint pathway. Recent results on posttranslational modifications and protein-protein interactions of some key factors provide new insights into the architecture of checkpoint protein complexes and their order of function.

The temperature-sensitive yeast DNA primase mutant pri1-M4 fails to execute an early step of DNA replication and exhibits a dominant, allele-specific sensitivity to DNA-damaging agents. pri1-M4 is defective in slowing down the rate of S phase progression and partially delaying the G1-S transition in response to DNA damage. Conversely, the G2 DNA damage response and the S-M checkpoint coupling completion of DNA replication to mitosis are unaffected. The signal transduction pathway leading to Rad53p phosphorylation induced by DNA damage is proficient in pri1-M4, and cell cycle delay caused by Rad53p overexpression is counteracted by the pri1-M4 mutation. Altogether, our results suggest that DNA primase plays an essential role in a subset of the Rad53p-dependent checkpoint pathways controlling cell cycle progression in response to DNA damage.

In this report we study the regulation of premeiotic DNA synthesis in Saccharomyces cerevisiae. DNA replication was monitored by fluorescence-activated cell sorting analysis and by analyzing the pattern of expression of the DNA polymerase alpha-primase complex. Wild-type cells and cells lacking one of the two principal regulators of meiosis, Ime1 and Ime2, were compared. We show that premeiotic DNA synthesis does not occur in ime1 delta diploids, but does occur in ime2 delta diploids with an 8-9 h delay. At late meiotic times, ime2 delta diploids exhibit an additional round of DNA synthesis. Furthermore, we show that in wild-type cells the B-subunit of DNA polymerase alpha is phosphorylated during premeiotic DNA synthesis, a phenomenon that has previously been reported for the mitotic cell cycle. Moreover, the catalytic subunit and the B-subunit of DNA polymerase alpha are specifically degraded during spore formation. Phosphorylation of the B-subunit does not occur in ime1 delta diploids, but does occur in ime2 delta diploids with an 8-9 h delay. In addition, we show that Ime2 is not absolutely required for commitment to meiotic recombination, spindle formation and nuclear division, although it is required for spore formation.

SPK1/RAD53/MEC2/SAD1 of Saccharomyces cerevisiae encodes an essential protein kinase that is required for activation of replication-sensitive and DNA damage-sensitive checkpoint arrest. We have investigated the regulation of phosphorylation and kinase activity of Spk1p during the cell cycle and by conditions that activate checkpoint pathways. Phosphorylation of Spk1p is induced by treatment of cells with agents that damage DNA or interfere with DNA synthesis. Although only S- and G2-phase cdc mutants arrest with hyperphosphorylated Spk1p, damage-induced phosphorylation of Spk1p can occur in G1 and M as well. Hydroxyurea (HU) induces phosphorylation of kinase-defective forms of Spk1p, demonstrating that this regulated phosphorylation of Spk1p occurs in trans. HU-induced phosphorylation is associated with increased catalytic activity of Spk1p. Furthermore, overexpression of wild-type SPK1, but not checkpoint-defective alleles, delays progression through the G1/S boundary. Damage-dependent phosphorylation of Spk1p requires both MEC1 and MEC3, whereas MEC1 but not MEC3, is required for replication block-induced phosphorylation. These data support the model that Spk1p is an essential intermediate component in a signal transduction pathway coupling damage and checkpoint functions to cell cycle arrest. This regulation is mediated through a protein kinase cascade that potentially includes Mec1p and Tel1p as the upstream kinases.

The yeast DNA polymerase alpha-primase B subunit functions in initiation of DNA replication. This protein is present in two forms, of 86 and 91 kDa, and the p91 polypeptide results from cell cycle-regulated phosphorylation of p86. The B subunit present in G1 arises by dephosphorylation of p91 while cells are exiting from mitosis, becomes phosphorylated in early S phase, and is competent and sufficient to initiate DNA replication. The B subunit transiently synthesized as a consequence of periodic transcription of the POL12 gene is phosphorylated no earlier than G2. Phosphorylation of the B subunit does not require execution of the CDC7-dependent step and ongoing DNA synthesis. We suggest that posttranslational modifications of the B subunit might modulate the role of DNA polymerase alpha-primase in DNA replication.

The four-subunit DNA polymerase alpha-primase complex is unique in its ability to synthesize DNA chains de novo, and some in vitro data suggest its involvement in initiation and elongation of chromosomal DNA replication, although direct in vivo evidence for a role in the initiation reaction is still lacking. The function of the B subunit of the complex is unknown, but the Saccharomyces cerevisiae POL12 gene, which encodes this protein, is essential for cell viability. We have produced different pol12 alleles by in vitro mutagenesis of the cloned gene. The in vivo analysis of our 18 pol12 alleles indicates that the conserved carboxy-terminal two-thirds of the protein contains regions that are essential for cell viability, while the more divergent NH2-terminal portion is partially dispensable. The characterization of the temperature-sensitive pol12-T9 mutant allele demonstrates that the B subunit is required for in vivo DNA synthesis and correct progression through S phase. Moreover, reciprocal shift experiments indicate that the POL12 gene product plays an essential role at the early stage of chromosomal DNA replication, before the hydroxyurea-sensitive step. A model for the role of the B subunit in initiation of DNA replication at an origin is presented.

The POL1 gene, encoding DNA polymerase alpha (pol alpha) in Saccharomyces cerevisiae, is transiently transcribed during the cell cycle at the G1/S phase boundary. Here we show that yeast pol alpha is present at every stage of the cell cycle, and its level only slightly increases following the peak of POL1 transcription. POL1 mRNA synthesis driven by a GAL1 promoter can be completely abolished without affecting the growth rate of logarithmically growing yeast cultures for several cell divisions, although the amount of the pol alpha polypeptide drops below the physiological level. Moreover, alpha-factor-arrested cells can enter S phase and divide synchronously even if POL1 transcription is abolished. These results indicate that the level of yeast pol alpha is not rate limiting and de novo synthesis of the enzyme is not required for entrance into S phase.

Phosphorylation of eukaryotic translation initiation factor 2 (eIF-2) in amino acid-starved cells of the yeast Saccharomyces cerevisiae reduces general protein synthesis but specifically stimulates translation of GCN4 mRNA. This regulatory mechanism is dependent on the nonessential GCN3 protein and multiple essential proteins encoded by GCD genes. Previous genetic and biochemical experiments led to the conclusion that GCD1, GCD2, and GCN3 are components of the GCD complex, recently shown to be the yeast equivalent of the mammalian guanine nucleotide exchange factor for eIF-2, known as eIF-2B. In this report, we identify new constituents of the GCD-eIF-2B complex and probe interactions between its different subunits. Biochemical evidence is presented that GCN3 is an integral component of the GCD-eIF-2B complex that, while dispensable, can be mutationally altered to have a substantial inhibitory effect on general translation initiation. The amino acid sequence changes for three gcd2 mutations have been determined, and we describe several examples of mutual suppression involving the gcd2 mutations and particular alleles of GCN3. These allele-specific interactions have led us to propose that GCN3 and GCD2 directly interact in the GCD-eIF-2B complex. Genetic evidence that GCD6 and GCD7 encode additional subunits of the GCD-eIF-2B complex was provided by the fact that reduced-function mutations in these genes are lethal in strains deleted for GCN3, the same interaction described previously for mutations in GCD1 and GCD2. Biochemical experiments showing that GCD6 and GCD7 copurify and coimmunoprecipitate with GCD1, GCD2, GCN3, and subunits of eIF-2 have confirmed that GCD6 and GCD7 are subunits of the GCD-eIF-2B complex. The fact that all five subunits of yeast eIF-2B were first identified as translational regulators of GCN4 strongly suggests that regulation of guanine nucleotide exchange on eIF-2 is a key control point for translation in yeast cells just as in mammalian cells.

The monoclonal antibody (mAb) 21A6, which specifically inhibits yeast DNA primase activity, has been used to verify whether only one of the two polypeptides of heterodimeric DNA primase (48 and 58 kDa) was responsible for DNA primase function in vitro. Immunoaffinity chromatography of a crude extract from cells of Saccharomyces cerevisiae on a mAb 21A6 protein A-Sepharose 6B column allowed the purification of the p48 primase polypeptide in an isolated form. This polypeptide was not derived through the dissociation of the four-subunit DNA polymerase alpha-primase complex, which can be purified from the same extract by affinity chromatography with a mAb recognizing the DNA polymerase alpha polypeptide. Therefore, free p48 was already present in the yeast extract and, possibly, within the cell. Isolated p48, devoid of any detectable p58 subunit, was sufficient for RNA primer synthesis, although free primase appeared to extend RNA primer-monomers to primer-multimers less efficiently. Primase activity associated with free p48 was highly unstable, indicating that although p48 bears the catalytic site, its association with the other polypeptides of the polymerase-primase complex plays an important role in stabilizing enzyme activity.

GCN4 is a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae whose expression is regulated by amino-acid availability at the translational level. GCD1 and GCD2 are negative regulators required for the repression of GCN4 translation under nonstarvation conditions that is mediated by upstream open reading frames (uORFs) in the leader of GCN4 mRNA. GCD factors are thought to be antagonized by the positive regulators GCN1, GCN2 and GCN3 in amino acid-starved cells to allow for increased GCN4 protein synthesis. Previous genetic studies suggested that GCD1, GCD2, and GCN3 have closely related functions in the regulation of GCN4 expression that involve translation initiation factor 2 (eIF-2). In agreement with these predictions, we show that GCD1, GCD2, and GCN3 are integral components of a high-molecular-weight complex of approximately 600,000 Da. The three proteins copurified through several biochemical fractionation steps and could be coimmunoprecipitated by using antibodies against GCD1 or GCD2. Interestingly, a portion of the eIF-2 present in cell extracts also cofractionated and coimmunoprecipitated with these regulatory proteins but was dissociated from the GCD1/GCD2/GCN3 complex by 0.5 M KCl. Incubation of a temperature-sensitive gcdl-101 mutant at the restrictive temperature led to a rapid reduction in the average size and quantity of polysomes, plus an accumulation of inactive 80S ribosomal couples; in addition, excess amounts of eIF-2 alpha, GCD1, GCD2, and GCN3 were found comigrating with free 40S ribosomal subunits. These results suggest that GCD1 is required for an essential function involving eIF-2 at a late step in the translation initiation cycle. We propose that lowering the function of this high-molecular-weight complex, or of eIF-2 itself, in amino acid-starved cells leads to reduced ribosomal recognition of the uORFs and increased translation initiation at the GCN4 start codon. Our results provide new insights into how general initiation factors can be regulated to affect gene-specific translational control.

The GCD2 protein is a translational repressor of GCN4, the transcriptional activator of multiple amino acid biosynthetic genes in Saccharomyces cerevisiae. We present evidence that GCD2 has a general function in the initiation of protein synthesis in addition to its gene-specific role in translational control of GCN4 expression. Two temperature-sensitive lethal gcd2 mutations result in sensitivity to inhibitors of protein synthesis at the permissive temperature, and the gcd2-503 mutation leads to reduced incorporation of labeled leucine into total protein following a shift to the restrictive temperature of 36 degrees C. The gcd2-503 mutation also results in polysome runoff, accumulation of inactive 80S ribosomal couples, and accumulation of at least one of the subunits of the general translation initiation factor 2 (eIF-2 alpha) in 43S-48S particles following a shift to the restrictive temperature. The gcd2-502 mutation causes accumulation of 40S subunits in polysomes, known as halfmers, that are indicative of reduced 40S-60S subunit joining at the initiation codon. These phenotypes suggest that GCD2 functions in the translation initiation pathway at a step following the binding of eIF-2.GTP.Met-tRNA(iMet) to 40S ribosomal subunits. consistent with this hypothesis, we found that inhibiting 40S-60S subunit joining by deleting one copy (RPL16B) of the duplicated gene encoding the 60S ribosomal protein L16 qualitatively mimics the phenotype of gcd2 mutations in causing derepression of GCN4 expression under nonstarvation conditions. However, deletion of RPL16B also prevents efficient derepression of GCN4 under starvation conditions, indicating that lowering the concentration of 60S subunits and reducing GCD2 function affect translation initiation at GCN4 in different ways. This distinction is in accord with a recently proposed model for GCN4 translational control in which ribosomal reinitiation at short upstream open reading frames in the leader of GCN4 mRNA is suppressed under amino acid starvation conditions to allow for increased reinitiation at the GCN4 start codon.

DNA primase activity of the yeast DNA polymerase-primase complex is related to two polypeptides, p58 and p48. The reciprocal role of these protein species has not yet been clarified, although both participate in formation of the active center of the enzyme. The gene encoding the p58 subunit has been cloned by screening of a lambda gt11 yeast genomic DNA library, using specific anti-p58 antiserum. Antibodies that inhibited DNA primase activity could be purified by lysates of Escherichia coli cells infected with a recombinant bacteriophage containing the entire gene, which we designate PR12. The gene was found to be transcribed in a 1.7-kilobase mRNA whose level appeared to fluctuate during the mitotic cell cycle. Nucleotide sequence determination indicated that PR12 encodes a 528-amino-acid polypeptide with a calculated molecular weight of 62,262. The gene is unique in the haploid yeast genome, and its product is essential for cell viability, as has been shown for other components of the yeast DNA polymerase-primase complex.

A highly selective affinity labeling procedure has been applied to map the active center of DNA primase from the yeast Saccharomyces cerevisiae. Enzyme molecules that have been modified by covalent attachment of benzaldehyde derivatives of adenine nucleotides are autocatalytically labeled by incubation with a radioactive ribonucleoside triphosphate. The affinity labeling of primase requires a template DNA, is not affected by DNase and RNase treatments, but is sensitive to proteinase K. Both the p58 and p48 subunits of yeast DNA primase appear to participate in the formation of the catalytic site of the enzyme, although UV-photocross-linking with [alpha-32P]ATP locates the ribonucleoside triphosphate binding site exclusively on the p48 polypeptide. The fixation of the radioactive product has been carried out also after the enzymatic reaction. Under this condition the RNA primers synthesized by the DNA polymerase-primase complex under uncoupled DNA synthesis conditions are linked to both DNA primase and DNA polymerase. When DNA synthesis is allowed to proceed first, the labeled RNA chains are fixed exclusively to the DNA polymerase polypeptide. These results, in accord with previous data, have been used to propose a model illustrating the interactions and the putative roles of the polypeptides of the DNA polymerase-primase complex.

The immunopurified yeast DNA polymerase--DNA primase complex is constituted by DNA polymerase I polypeptides and by three other protein species, called p74, p58 and p48, which we show to be immunologically unrelated. The gene encoding the p48 polypeptide has been identified by immunological screening of a lambda gt11 yeast genomic DNA library. Antiserum specific for p48 inhibits DNA primase, and immunoreactive, inhibitory antibodies are affinity-purified by the clone-encoded protein, thus relating the p48 polypeptide to DNA primase activity. The entire gene has been cloned, and the 1.45-kb p48 mRNA is overproduced in cells containing the gene in high copy number. Gene disruption and Southern hybridization experiments demonstrate that the p48 protein is encoded by a single gene and it performs an essential function.

The yeast DNA polymerase-primase complex is composed of four polypeptides designated p180, p74, p58 and p48. All the genes coding for these polypeptides have now been cloned. By protein sequence comparison we found that yeast DNA polymerase I (alpha) shares three major regions of homology with several DNA polymerases. A fourth region, called region P, is conserved in yeast and human DNA polymerase alpha. The site of a temperature-sensitive mutation in the POL1 gene which causes decreased stability of the polymerase-primase complex has been sequenced and falls in this region. We hypothesize that region P is important for protein-protein interactions. Highly selective biochemical methods might be similarly important to distinguish functional domains in the polymerase-primase complex. An autocatalytic affinity labeling procedure has been applied to map the active center of yeast DNA primase. From this approach we conclude that both primase subunits (p48 and p58) participate in the formation of the catalytic site of the enzyme.