Mitotic DNA damage and replication checkpoints in yeast

Studies of the genetics of G₂/M checkpoints in budding and fission yeasts have produced many of the defining concepts of checkpoint biology. Recent progress in the biochemistry of the checkpoint gene products is adding a mechanistic understanding to our models and identifying the components of the normal cell cycle machinery that are targeted by checkpoints.     

Telomeres and DNA damage checkpoints

In all eukaryotic organisms, interruptions in duplex DNA molecules elicit a DNA damage response, which includes activation of DNA repair machineries and surveillance mechanisms, known as DNA damage checkpoints. Telomeres and double-strand breaks (DSBs) share the common feature of being physical ends of chromosomes. However, unlike DSBs, telomeres do not activate the DNA damage checkpoints and are usually protected from end-to-end fusions and other processing events that normally promote repair of DNA breaks. This indicates that they are shielded from being recognized and processed as DSBs. On the other hand, chromosome ends resemble damaged DNA, as several factors required for DNA repair and checkpoint networks play important roles in telomere length maintenance. Due to the critical role of both DNA damage checkpoints and telomere homeostasis in maintaining genetic stability and in counteracting cancer development, the knowledge of their interconnections is essential for our understanding of these key cellular controls.     

Rad4TopBP1, a scaffold protein, plays separate roles in DNA damage and replication checkpoints and DNA replication

Rad4TopBP1, a BRCT domain protein, is required for both DNA replication and checkpoint responses. Little is known about how the multiple roles of Rad4TopBP1 are coordinated in maintaining genome integrity. We show here that Rad4TopBP1 of fission yeast physically interacts with the checkpoint sensor proteins, the replicative DNA polymerases, and a WD-repeat protein, Crb3. We identified four novel mutants to investigate how Rad4TopBP1 could have multiple roles in maintaining genomic integrity. A novel mutation in the third BRCT domain of rad4+TopBP1 abolishes DNA damage checkpoint response, but not DNA replication, replication checkpoint, and cell cycle progression. This mutant protein is able to associate with all three replicative polymerases and checkpoint proteins Rad3ATR-Rad26ATRIP, Hus1, Rad9, and Rad17 but has a compromised association with Crb3. Furthermore, the damaged-induced Rad9 phosphorylation is significantly reduced in this rad4TopBP1 mutant. Genetic and biochemical analyses suggest that Crb3 has a role in the maintenance of DNA damage checkpoint and influences the Rad4TopBP1 damage checkpoint function. Taken together, our data suggest that Rad4TopBP1 provides a scaffold to a large complex containing checkpoint and replication proteins thereby separately enforcing checkpoint responses to DNA damage and replication perturbations during the cell cycle.     

Induction by adozelesin and hydroxyurea of origin recognition complex-dependent DNA damage and DNA replication checkpoints in Saccharomyces cerevisiae

DNA damaging agents induce a conserved intra-S-phase checkpoint that inhibits DNA replication in eukaryotic cells. To better understand this checkpoint and its role in determining the efficacy of antitumor drugs that damage DNA, we examined the effects of adozelesin, a DNA-alkylating antitumor agent that has a profound inhibitory effect on initiation of DNA replication in mammals, on the replication of Saccharomyces cerevisiae chromosomes. Adozelesin inhibited initiation of S. cerevisiae DNA replication by inducing an intra-S-phase DNA damage checkpoint. This inhibitory effect was abrogated in orc2-1 cells containing a temperature-sensitive mutation in a component of the origin recognition complex (ORC) that also causes a defect in initiation. The orc2-1 mutation also caused a defect in a checkpoint that regulates the activation of origins in late S phase in cells treated with hydroxyurea. Defects in both initiation and checkpoint regulation in the orc2-1 strain were suppressed by deletion of a gene encoding a putative acetyltransferase, SAS2. Adozelesin also induced a cellular response that requires a function of ORC in G(1). A similar G(1)-specific response in mammals may contribute to the cytotoxic and antitumor properties of this and other DNA-damaging drugs.     

Differential effects of caffeine on DNA damage and replication cell cycle checkpoints in the fission yeast Schizosaccharomyces pombe

Caffeine potentiates the lethal effects of ultraviolet and ionising radiation on wild-type Schizosaccharomyces pombe cells. In previous studies this was attributed to the inhibition by caffeine of a novel DNA repair pathway in S. pombe that was absent in the budding yeast Saccharomyces cerevisiae. Studies with radiation-sensitive S. pombe mutants suggested that this caffeine-sensitive pathway could repair ultraviolet radiation damage in the absence of nucleotide excision repair. The alternative pathway was thought to be recombinational and to operate in the G₂ phase of the cell cycle. However, in this study we show that cells held in G₁ of the cell cycle can remove ultraviolet-induced lesions in the absence of nucleotide excision repair. We also show that recombination-defective mutants, and those now known to define the alternative repair pathway, still exhibit the caffeine effect. Our observations suggest that the basis of the caffeine effect is not due to direct inhibition of recombinational repair. The mutants originally thought to be involved in a caffeine-sensitive recombinational repair process are now known to be defective in arresting the cell cycle in S and/or G₂ following DNA damage or incomplete replication. The gene products may also have an additional role in a DNA repair or damage tolerance pathway. The effect of caffeine could, therefore, be due to interference with DNA damage checkpoints, or inhibition of the DNA damage repair/tolerance pathway. Using a combination of flow cytometric analysis, mitotic index analysis and fluorescence microscopy we show that caffeine interferes with intra-S phase and G₂ DNA damage checkpoints, overcoming cell cycle delays associated with damaged DNA. In contrast, caffeine has no effect on the DNA replication S phase checkpoint in reponse to inhibition of DNA synthesis by hydroxyurea.     

Linking DNA damage to cell cycle checkpoints

Cell cycle checkpoints constitute a network of signal transduction mechanisms to monitor DNA damage and replication and thereby regulate progression through the cell cycle. A series of events is triggered in cells upon DNA damage. Here we describe a framework for the understanding of the functions of the core components involved in the cell cycle response to DNA damage and the relevance to the origin of cancer.     

Dual cell cycle checkpoints sensitive to chromosome replication and DNA damage in the budding yeast Saccharomyces cerevisiae.

In eucaryotic cells chromosomes must be fully replicated and repaired before mitosis begins. Genetic studies indicate that this dependence of mitosis on completion of DNA replication and DNA repair derives from a negative control called a checkpoint which somehow checks for replication and DNA damage and blocks cell entry into mitosis. Here we summarize our current understanding of the genetic components of the cell cycle checkpoint in budding yeast. Mutants were identified and their phase and signal specificity tested primarily through interactions of the arrest-defective mutants with cell division cycle mutants. The results indicate that dual checkpoint controls exist in budding yeast, one control sensitive to inhibition of DNA replication (S-phase checkpoint), and a distinct but overlapping control sensitive to DNA repair (G2 checkpoint). Six genes are required for arrest in G2 phase after DNA damage (RAD9, RAD17, RAD24, MEC1, MEC2, and MEC3), and two of these are also essential for arrest in S phase when DNA replication is blocked (MEC1 and MEC2).     

Rfc4 interacts with Rpa1 and is required for both DNA replication and DNA damage checkpoints in Saccharomyces cerevisiae

The large subunit of replication protein A (Rpa1) consists of three single-stranded DNA binding domains and an N-terminal domain (Rpa1N) of unknown function. To determine the essential role of this domain we searched for mutations that require wild-type Rpa1N for viability in yeast. A mutation in RFC4, encoding a small subunit of replication factor C (RFC), was found to display allele-specific interactions with mutations in the gene encoding Rpa1 (RFA1). Mutations that map to Rpa1N and confer sensitivity to the DNA synthesis inhibitor hydroxyurea, such as rfa1-t11, are lethal in combination with rfc4-2. The rfc4-2 mutant itself is sensitive to hydroxyurea, and like rfc2 and rfc5 strains, it exhibits defects in the DNA replication block and intra-S checkpoints. RFC4 and the DNA damage checkpoint gene RAD24 were found to be epistatic with respect to DNA damage sensitivity. We show that the rfc4-2 mutant is defective in the G(1)/S DNA damage checkpoint response and that both the rfc4-2 and rfa1-t11 strains are defective in the G(2)/M DNA damage checkpoint. Thus, in addition to its essential role as part of the clamp loader in DNA replication, Rfc4 plays a role as a sensor in multiple DNA checkpoint pathways. Our results suggest that a physical interaction between Rfc4 and Rpa1N is required for both roles.     

DNA damage checkpoints are involved in postreplication repair

Saccharomyces cerevisiae MMS2 encodes a ubiquitin-conjugating enzyme variant, belongs to the error-free branch of the RAD6 postreplication repair (PRR) pathway, and is parallel to the REV3-mediated mutagenesis branch. A mutation in genes of either the MMS2 or the REV3 branch does not result in extreme sensitivity to DNA-damaging agents; however, deletion of both subpathways of PRR results in a synergistic phenotype. Nevertheless, the double mutant is not as sensitive to DNA-damaging agents as a rad6 or rad18 mutant defective in the entire PRR pathway, suggesting the presence of an additional subpathway within PRR. A synthetic lethal screen was employed in the presence of a sublethal dose of a DNA-damaging agent to identify novel genes involved in PRR, which resulted in the isolation of RAD9 as a candidate PRR gene. Epistatic analysis showed that rad9 is synergistic to both mms2 and rev3 with respect to killing by methyl methanesulfonate (MMS), and the triple mutant is nearly as sensitive as the rad18 single mutant. In addition, rad9 rad18 is no more sensitive to MMS than the rad18 single mutant, suggesting that rad9 plays a role within the PRR pathway. Moreover, deletion of RAD9 reduces damage-induced mutagenesis and the mms2 spontaneous and induced mutagenesis is partially dependent on the RAD9 gene. We further demonstrated that the observed synergistic interactions apply to any two members between different branches of PRR and G₁/S and G₂/M checkpoint genes. These results suggest that a damage checkpoint is essential for tolerance mediated by both the error-free and error-prone branches of PRR.     

Roles of the mitotic inhibitors Wee1 and Mik1 in the G(2) DNA damage and replication checkpoints

The G(2) DNA damage and DNA replication checkpoints in many organisms act through the inhibitory phosphorylation of Cdc2 on tyrosine-15. This phosphorylation is catalyzed by the Wee1/Mik1 family of kinases. However, the in vivo role of these kinases in checkpoint regulation has been unclear. We show that, in the fission yeast Schizosaccharomyces pombe, Mik1 is a target of both checkpoints and that the regulation of Mik1 is, on its own, sufficient to delay mitosis in response to the checkpoints. Mik1 appears to have two roles in the DNA damage checkpoint; one in the establishment of the checkpoint and another in its maintenance. In contrast, Wee1 does not appear to be involved in the establishment of either checkpoint.     

Differential effects of caffeine on DNA damage and replication cell cycle checkpoints in the fission yeast Schizosaccharomyces pombe

Caffeine potentiates the lethal effects of ultraviolet and ionising radiation on wild-type Schizosaccharomyces pombe cells. In previous studies this was attributed to the inhibition by caffeine of a novel DNA repair pathway in S. pombe that was absent in the budding yeast Saccharomyces cerevisiae. Studies with radiation-sensitive S. pombe mutants suggested that this caffeine-sensitive pathway could repair ultraviolet radiation damage in the absence of nucleotide excision repair. The alternative pathway was thought to be recombinational and to operate in the G₂ phase of the cell cycle. However, in this study we show that cells held in G₁ of the cell cycle can remove ultraviolet-induced lesions in the absence of nucleotide excision repair. We also show that recombination-defective mutants, and those now known to define the alternative repair pathway, still exhibit the caffeine effect. Our observations suggest that the basis of the caffeine effect is not due to direct inhibition of recombinational repair. The mutants originally thought to be involved in a caffeine-sensitive recombinational repair process are now known to be defective in arresting the cell cycle in S and/or G₂ following DNA damage or incomplete replication. The gene products may also have an additional role in a DNA repair or damage tolerance pathway. The effect of caffeine could, therefore, be due to interference with DNA damage checkpoints, or inhibition of the DNA damage repair/tolerance pathway. Using a combination of flow cytometric analysis, mitotic index analysis and fluorescence microscopy we show that caffeine interferes with intra-S phase and G₂ DNA damage checkpoints, overcoming cell cycle delays associated with damaged DNA. In contrast, caffeine has no effect on the DNA replication S phase checkpoint in reponse to inhibition of DNA synthesis by hydroxyurea. Received: 16 June 1998 / Accepted: 13 July 1998     

Molecular anatomy of the human excision nuclease assembled at sites of DNA damage

Human nucleotide excision repair is initiated by six repair factors (XPA, RPA, XPC-HR23B, TFIIH, XPF-ERCC1, and XPG) which sequentially assemble at sites of DNA damage and effect excision of damage-containing oligonucleotides. We here describe the molecular anatomy of the human excision nuclease assembled at the site of a psoralen-adducted thymine. Three polypeptides, primarily positioned 5' to the damage, are in close physical proximity to the psoralen lesion and thus are cross-linked to the damaged DNA: these proteins are RPA70, RPA32, and the XPD subunit of TFIIH. While both XPA and XPC bind damaged DNA and are required for XPD cross-linking to the psoralen-adducted base, neither XPA nor XPC is cross-linked to the psoralen adduct. The presence of other repair factors, in particular TFIIH, alters the mode of RPA binding and the position of its subunits relative to the psoralen lesion. Based on these results, we propose that RPA70 makes the initial contact with psoralen-damaged DNA but that within preincision complexes, it is RPA32 and XPD that are in close contact with the lesion.     

When heat casts a spell on the DNA damage checkpoints

Peregrine Laziosi (1265–1345), an Italian priest, became the patron saint of cancer patients when the tumour in his left leg miraculously disappeared after he developed a fever. Elevated body temperature can cause tumours to regress and sensitizes cancer cells to agents that break DNA. Why hyperthermia blocks the repair of broken chromosomes by changing the way that the DNA damage checkpoint kinases ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) are activated is an unanswered question. This review discusses the current knowledge of how heat affects the ATR–Chk1 and ATM–Chk2 kinase networks, and provides a possible explanation of why homeothermal organisms such as humans still possess this ancient heat response.     

Activation of DNA damage checkpoints in CHO cells requires a certain level of DNA damage

DNA damage activates checkpoint controls in eukaryotic cells. It is not clear, however, whether a certain level of DNA damage is required for the activation of DNA damage checkpoints. We show here that low levels of DNA damage in Chinese hamster ovary (CHO) cells induced by short exposure to hydroxyurea (HU) did not trigger checkpoints, whereas higher levels of DNA damage caused by longer exposure to HU resulted in a cell cycle arrest. Our results argue that a threshold of DNA damage is necessary for activation of DNA damage checkpoints.     

DNA damage checkpoints in stem cells, ageing and cancer

DNA damage induces cell-intrinsic checkpoints, including p53 and retinoblastoma (RB), as well as upstream regulators (exonuclease 1 (EXO1), ataxia telangiectasia mutated (ATM), ATR, p16(INK4a) and p19(ARF)) and downstream targets (p21, PUMA (p53 upregulated modulator of apoptosis) and sestrins). Clearance of damaged cells by cell-intrinsic checkpoints suppresses carcinogenesis but as a downside may impair stem cell and tissue maintenance during ageing. Modulating the activity of DNA damage checkpoints can either accelerate or decelerate tissue ageing and age-related carcinogenesis. The outcome depends on cell-intrinsic and cell-extrinsic mechanisms that regulate the clearance of damaged cells and on the molecular context in ageing tissues, including the level of DNA damage accumulation itself.