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RNA:DNA hybrids are transient physiological intermediates that arise during several cellular processes such as DNA replication. In pathological situations, they may stably accumulate and pose a threat to genome integrity. Cellular RNase H activities process these structures to restore the correct DNA:DNA sequence. Yeast cells lacking RNase H are negatively affected by depletion of deoxyribonucleotide pools necessary for DNA replication. Here we show that the translesion synthesis DNA polymerase η (Pol η) plays a role in DNA replication under low deoxyribonucleotides condition triggered by hydroxyurea. In particular, the catalytic reaction performed by Pol η is detrimental for RNase H deficient cells, causing DNA damage checkpoint activation and G2/M arrest. Moreover, a Pol η mutant allele with enhanced ribonucleotide incorporation further exacerbates the sensitivity to hydroxyurea of cells lacking RNase H activities. Our data are compatible with a model in which Pol η activity facilitates the formation or stabilization of RNA:DNA hybrids at stalled replication forks. However, in a scenario where RNase H activity fails to restore DNA, these hybrids become highly toxic for cells., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The chemical identity and integrity of the genome is challenged by the incorporation of ribonucleoside triphosphates (rNTPs) in place of deoxyribonucleoside triphosphates (dNTPs) during replication. Misincorporation is limited by the selectivity of DNA replicases. We show that accumulation of ribonucleoside monophosphates (rNMPs) in the genome causes replication stress and has toxic consequences, particularly in the absence of RNase H1 and RNase H2, which remove rNMPs. We demonstrate that postreplication repair (PRR) pathways-MMS2-dependent template switch and Pol ζ-dependent bypass-are crucial for tolerating the presence of rNMPs in the chromosomes; indeed, we show that Pol ζ efficiently replicates over 1-4 rNMPs. Moreover, cells lacking RNase H accumulate mono- and polyubiquitylated PCNA and have a constitutively activated PRR. Our findings describe a crucial function for RNase H1, RNase H2, template switch, and translesion DNA synthesis in overcoming rNTPs misincorporated during DNA replication, and may be relevant for the pathogenesis of Aicardi-Goutières syndrome., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Replication fork integrity, which is essential for the maintenance of genome stability, is monitored by checkpoint-mediated phosphorylation events. 14-3-3 proteins are able to bind phosphorylated proteins and were shown to play an undefined role under DNA replication stress. Exonuclease 1 (Exo1) processes stalled replication forks in checkpoint-defective yeast cells. We now identify 14-3-3 proteins as in vivo interaction partners of Exo1, both in yeast and mammalian cells. Yeast 14-3-3-deficient cells fail to induce Mec1-dependent Exo1 hyperphosphorylation and accumulate Exo1-dependent ssDNA gaps at stalled forks, as revealed by electron microscopy. This leads to persistent checkpoint activation and exacerbated recovery defects. Moreover, using DNA bi-dimensional electrophoresis, we show that 14-3-3 proteins promote fork progression under limiting nucleotide concentrations. We propose that 14-3-3 proteins assist in controlling the phosphorylation status of Exo1 and additional unknown targets, promoting fork progression, stability, and restart in response to DNA replication stress., Competing Interests: The authors have declared that no competing interests exist.

Ultraviolet (UV) light induces DNA-damage checkpoints and mutagenesis, which are involved in cancer protection and tumorigenesis, respectively. How cells identify DNA lesions and convert them to checkpoint-activating structures is a major question. We show that during repair of UV lesions in noncycling cells, Exo1-mediated processing of nucleotide excision repair (NER) intermediates competes with repair DNA synthesis. Impediments of the refilling reaction allow Exo1 to generate extended ssDNA gaps, detectable by electron microscopy, which drive Mec1 kinase activation and will be refilled by long-patch repair synthesis, as shown by DNA combing. We provide evidence that this mechanism may be stimulated by closely opposing UV lesions, represents a strategy to redirect problematic repair intermediates to alternative repair pathways, and may also be extended to physically different DNA damages. Our work has significant implications for understanding the coordination between repair of DNA lesions and checkpoint pathways to preserve genome stability., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Saccharomyces cerevisiae Rad9 is required for an effective DNA damage response throughout the cell cycle. Assembly of Rad9 on chromatin after DNA damage is promoted by histone modifications that create docking sites for Rad9 recruitment, allowing checkpoint activation. Rad53 phosphorylation is also dependent upon BRCT-directed Rad9 oligomerization; however, the crosstalk between these molecular determinants and their functional significance are poorly understood. Here we report that, in the G1 and M phases of the cell cycle, both constitutive and DNA damage-dependent Rad9 chromatin association require its BRCT domains. In G1 cells, GST or FKBP dimerization motifs can substitute to the BRCT domains for Rad9 chromatin binding and checkpoint function. Conversely, forced Rad9 dimerization in M phase fails to promote its recruitment onto DNA, although it supports Rad9 checkpoint function. In fact, a parallel pathway, independent on histone modifications and governed by CDK1 activity, allows checkpoint activation in the absence of Rad9 chromatin binding. CDK1-dependent phosphorylation of Rad9 on Ser11 leads to specific interaction with Dpb11, allowing Rad53 activation and bypassing the requirement for the histone branch., Competing Interests: The authors have declared that no competing interests exist.

Checkpoints are surveillance mechanisms that constitute a barrier to oncogenesis by preserving genome integrity. Loss of checkpoint function is an early event in tumorigenesis. Polo kinases (Plks) are fundamental regulators of cell cycle progression in all eukaryotes and are frequently overexpressed in tumors. Through their polo box domain, Plks target multiple substrates previously phosphorylated by CDKs and MAPKs. In response to DNA damage, Plks are temporally inhibited in order to maintain the checkpoint-dependent cell cycle block while their activity is required to silence the checkpoint response and resume cell cycle progression. Here, we report that, in budding yeast, overproduction of the Cdc5 polo kinase overrides the checkpoint signaling induced by double strand DNA breaks (DSBs), preventing the phosphorylation of several Mec1/ATR targets, including Ddc2/ATRIP, the checkpoint mediator Rad9, and the transducer kinase Rad53/CHK2. We also show that high levels of Cdc5 slow down DSB processing in a Rad9-dependent manner, but do not prevent the binding of checkpoint factors to a single DSB. Finally, we provide evidence that Sae2, the functional ortholog of human CtIP, which regulates DSB processing and inhibits checkpoint signaling, is regulated by Cdc5. We propose that Cdc5 interferes with the checkpoint response to DSBs acting at multiple levels in the signal transduction pathway and at an early step required to resect DSB ends., Competing Interests: The authors have declared that no competing interests exist.

Rad53 is an essential protein kinase governing DNA damage and replication stress checkpoints in budding yeast. It also appears to be involved in cellular morphogenesis processes. Mass spectrometry analyses revealed that Rad53 is phosphorylated at multiple SQ/TQ and at SP/TP residues, which are typical consensus sites for phosphatidylinositol 3-kinase-related kinases and CDKs, respectively. Here we show that Clb-CDK1 phosphorylates Rad53 at Ser(774) in metaphase. This phosphorylation event does not influence the DNA damage and replication checkpoint roles of Rad53, and it is independent of the spindle assembly checkpoint network. Moreover, the Ser-to-Asp mutation, mimicking a constitutive phosphorylation state at site 774, causes sensitivity to calcofluor, supporting a functional linkage between Rad53 and cellular morphogenesis.

Cells respond to DNA double-strand breaks (DSBs) and uncapped telomeres by recruiting checkpoint and repair factors to the site of lesions. Single-stranded DNA (ssDNA) is an important intermediate in the repair of DSBs and is produced also at uncapped telomeres. Here, we provide evidence that binding of the checkpoint protein Rad9, through its Tudor domain, to methylated histone H3-K79 inhibits resection at DSBs and uncapped telomeres. Loss of DOT1 or mutations in RAD9 influence a Rad50-dependent nuclease, leading to more rapid accumulation of ssDNA, and faster activation of the critical checkpoint kinase, Mec1. Moreover, deletion of RAD9 or DOT1 partially bypasses the requirement for CDK1 in DSB resection. Interestingly, Dot1 contributes to checkpoint activation in response to low levels of telomere uncapping but is not essential with high levels of uncapping. We suggest that both Rad9 and histone H3 methylation allow transmission of the damage signal to checkpoint kinases, and keep resection of damaged DNA under control influencing, both positively and negatively, checkpoint cascades and contributing to a tightly controlled response to DNA damage.

Translesion DNA synthesis (TLS) is one of the mechanisms involved in lesion bypass during DNA replication. Three TLS polymerases (Pol) are present in the yeast Saccharomyces cerevisiae: Pol zeta, Pol eta and the product of the REV1 gene. Rev1 is considered a deoxycytidyl transferase because it almost exclusively inserts a C residue in front of the lesion. Even though REV1 is required for most of the UV-induced and spontaneous mutagenesis events, the role of Rev1 is poorly understood since its polymerase activity is often dispensable. Rev1 interacts with several TLS polymerases in mammalian cells and may act as a platform in the switching mechanism required to substitute a replicative polymerase with a TLS polymerase at the sites of DNA lesions. Here we show that yeast Rev1 is a phosphoprotein, and the level of this modification is cell cycle regulated under normal growing conditions. Rev1 is unphosphorylated in G1, starts to be modified while cells are passing S phase and it becomes hyper-phosphorylated in mitosis. Rev1 is also hyper-phosphorylated in response to a variety of DNA damaging agents, including treatment with a radiomimetic drug mostly causing double-strand breaks (DSB). By using the chromosome spreading technique we found the Rev1 is bound to chromosomes throughout the cell cycle, and its binding does not significantly increase in response to genotoxic stress. Therefore, Rev1 phosphorylation does not appear to modulate its binding to chromosomes, suggesting that such modification may influence other aspects of the TLS process. Rev1 binding under damaged and undamaged conditions, is at least partially dependent on MEC1, a gene playing a pivotal role in the DNA damage checkpoint cascade. This genetic dependency may suggest a role for MEC1 in spontaneous mutagenesis events, which require a functional REV1 gene.

Haspin is a protein kinase identified in mouse and human cells, and genes coding for haspin-like proteins are present in virtually all eukaryotic genomes sequenced so far. Two haspin homologues, called Alk1 and Alk2, are present in the yeast Saccharomyces cerevisiae. Both Alk1 and Alk2 exhibit a weak auto-kinase activity in vitro, are phosphoproteins in vivo and are hyperphosphorylated in response to DNA damage. The amount and modification of the two proteins is greatly regulated during the cell cycle. In fact, Alk1 and Alk2 levels peak in mitosis and late-S/G2, respectively, and phosphorylation of both proteins is maximal in mitosis. Control of protein stability plays a major role in Alk2 regulation. The half-life of Alk2 is particularly short in G1; mutagenesis and genetic analysis indicate that its degradation is controlled by the APC pathway. Overexpression of ALK2, but not of ALK1, causes a mitotic arrest, which is correlated to the kinase activity of the protein. This finding, together with its cell cycle regulation, suggests a role for Alk2 in the control of mitosis.

The use of translesion synthesis (TLS) polymerases to bypass DNA lesions during replication constitutes an important mechanism to restart blocked/stalled DNA replication forks. Because TLS polymerases generally have low fidelity on undamaged DNA, the cell must regulate the interaction of TLS polymerases with damaged versus undamaged DNA to maintain genome integrity. The Saccharomyces cerevisiae checkpoint proteins Ddc1, Rad17, and Mec3 form a clamp-like structure (the 9-1-1 clamp) that has physical similarity to the homotrimeric sliding clamp proliferating cell nuclear antigen, which interacts with and promotes the processivity of the replicative DNA polymerases. In this work, we demonstrate both an in vivo and in vitro physical interaction between the Mec3 and Ddc1 subunits of the 9-1-1 clamp and the Rev7 subunit of the Polzeta TLS polymerase. In addition, we demonstrate that loss of Mec3, Ddc1, or Rad17 results in a decrease in Polzeta-dependent spontaneous mutagenesis. These results suggest that, in addition to its checkpoint signaling role, the 9-1-1 clamp may physically regulate Polzeta-dependent mutagenesis by controlling the access of Polzeta to damaged DNA.

The DNA damage checkpoint is a surveillance mechanism activated by DNA lesions and devoted to the maintenance of genome stability. It is considered as a signal transduction cascade, involving a sensing step, the activation of a set of protein kinases and the transmission and amplification of the damage signal through several phosphorylation events. In budding yeast many players of this pathway have been identified. Recent work showed that G1 and G2 checkpoint activation in response to UV irradiation requires prior recognition and processing of UV lesions by nucleotide excision repair (NER) factors that likely recruit checkpoint proteins near the damage. However, another report suggested that NER was not required for checkpoint function. Since the functional relationship between repair mechanisms and checkpoint activation is a very important issue in the field, we analyzed, under different experimental conditions, whether lesion processing by NER is required for checkpoint activation. We found that DNA damage checkpoint can be triggered in an NER-independent manner only if cells are subjected to liquid holding after UV treatment. This incubation causes a time-dependent breakage of DNA strands in NER-deficient cells and leads to partial activation of the checkpoint kinase. The analysis of the genetic requirements for this alternative activation pathway suggest that it requires Mec1 and the Rad17 complex and that the observed DNA breaks are likely to be due to spontaneous decay of damaged DNA.

The mechanisms used by checkpoints to identify DNA lesions are poorly understood and may involve the function of repair proteins. Looking for mutants specifically defective in activating the checkpoint following UV lesions, but proficient in the response to methyl methane sulfonate and double-strand breaks, we isolated cdu1-1, which is allelic to RAD14, the homolog of human XPA, involved in lesion recognition during nucleotide excision repair (NER). Rad14 was also isolated as a partner of the Ddc1 checkpoint protein in a two-hybrid screening, and physical interaction was proven by co-immunoprecipitation. We show that lesion recognition is not sufficient for checkpoint activation, but processing, carried out by repair factors, is required for recruiting checkpoint proteins to damaged DNA. Mutations affecting the core NER machinery abolish G1 and G2 checkpoint responses to UV, preventing activation of the Mec1 kinase and its binding to chromosomes. Conversely, elimination of transcription-coupled or global genome repair alone does not affect checkpoints, suggesting a possible interpretation for the heterogeneity in cancer susceptibility observed in different NER syndrome patients.

Rad17-Mec3-Ddc1 forms a proliferating cell nuclear antigen-like complex that is required for the DNA damage response in Saccharomyces cerevisiae and acts at an early step of the signal transduction cascade activated by DNA lesions. We used the mec3-dn allele, which causes a dominant negative checkpoint defect in G1 but not in G2, to test the stability of the complex in vivo and to correlate its assembly and disassembly with the mechanisms controlling checkpoint activation. Under physiological conditions, the mutant complex is formed both in G1 and G2, although the mutant phenotype is detectable only in G1, suggesting that is not the presence of the mutant complex per se to cause a checkpoint defect. Our data indicate that the Rad17-Mec3-Ddc1 complex is very stable, and it takes several hours to replace Mec3 with Mec3-dn within a wild type complex. On the other hand, the mutant complex is rapidly assembled when starting from a condition where the complex is not pre-assembled, indicating that the critical factor for the substitution is the disassembly step rather than complex formation. Moreover, the kinetics of mutant complex assembly, starting from conditions in which the wild type form is present, parallels the kinetics of checkpoint inactivation, suggesting that the complex acts in a stoichiometric way, rather than catalytically.

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.

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 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.

We report the 9210 bp sequence from a segment of yeast chromosome III cloned from strain AB972 in lambda PM3270. Analysis of this sequence and its comparison with the one derived from the corresponding segment of strain XJ24-24A revealed that the AB972 region contains a duplication of about 2 kb and a Ty element, which are not found in XJ24-24A and cause a quite significant rearrangement of the whole region. We performed functional analysis of YCR28, the largest open reading frame we found in both AB972 and XJ24-24A. YCR28 encodes a putative protein of 512 amino acids with some similarities to yeast allontoate permease. Its disruption does not cause any detectable phenotype on rich medium or on allantoate medium, while we observed a strain-dependent effect on sensitivity to amino acid balance and to 3-aminotriazole, when cells were grown in synthetic medium.

Eukaryotic DNA primases are composed of two distinct subunits of 48-50 and 58-60 kDa. The amino acid sequences derived from the nucleotide sequences of the cloned genes are known only for the yeast and mouse polypeptides, and the extensive homology between the corresponding mouse and yeast subunits suggests conservation of functional domains. We were able to express in Saccharomyces cerevisiae the homologous and mouse primase-encoding genes under the control of both the constitutive ADH1 and the inducible GAL1 strong promoters, thus obtaining strains producing relevant amounts of the different polypeptides. In vivo complementation studies showed that neither one of the wild-type mouse primase-encoding genes was able to rescue the lethal or temperature-sensitive phenotype caused by mutations in the yeast PRI1 or PRI2 genes, indicating that these proteins, even if structurally and functionally very similar, might be involved in critical species-specific interactions during DNA replication.

We have analyzed the effects of temperature-sensitivity (ts)-conferring mutations in the Saccharomyces cerevisiae DNA polymerase I-encoding gene on cell growth, in vivo DNA synthesis, intrachromosomal gene conversion and pop-out recombination. Also, we have identified the molecular defect responsible for the ts phenotype. Two mutant alleles (cdc17-1, cdc17-2) were originally identified as cell-cycle mutations, while a third mutation (hpr3) was found during a genetic screening for mutants with a hyper-recombination phenotype. Both cdc17-2 and hpr3 cells complete one round of cell division and DNA replication after shift to nonpermissive temperature, before being arrested as dumbbell-shaped cells. Conversely, the cdc17-1 mutation immediately blocks growth and DNA synthesis at 37 degrees C. No substantial difference was observed in the frequency of intrachromosomal gene conversion and pop-out recombination events, when hpr3 and cdc17-1 were compared to the previously characterized pol1-1 mutant. These two frequencies were ten- to 30-fold above wild-type level at semipermissive temperature. In each mutant, a single bp substitution, causing the replacement of Gly residues by either Asp (cdc17-1, cdc17-2) or Glu (hpr3) in yeast DNA polymerase I is responsible for the ts phenotype.

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.