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.     

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.     

Telomeres are double-strand DNA breaks hidden from DNA damage responses

A network of ATM/ATR-mediated events regulates cell cycle checkpoints and genomic integrity and contributes to the processing of DNA double-strand breaks in both genomic DNA and at telomeres. In yeast and in human cells, investigators, including, and Herbig et al., published in this issue of Molecular Cell, are beginning to decipher the signaling pathways involved at the telomeres.     

Molecular anatomy of the DNA damage and replication checkpoints

Cell cycle checkpoints are signal transduction pathways that enforce the orderly execution of the cell division cycle and arrest the cell cycle upon the occurrence of undesirable events, such as DNA damage, replication stress, and spindle disruption. The primary function of the cell cycle checkpoint is to ensure that the integrity of chromosomal DNA is maintained. DNA lesions and disrupted replication forks are thought to be recognized by the DNA damage checkpoint and replication checkpoint, respectively. Both checkpoints initiate protein kinase-based signal transduction cascade to activate downstream effectors that elicit cell cycle arrest, DNA repair, or apoptosis that is often dependent on dose and cell type. These actions prevent the conversion of aberrant DNA structures into inheritable mutations and minimize the survival of cells with unrepairable damage. Genetic components of the damage and replication checkpoints have been identified in yeast and humans, and a working model is beginning to emerge. We summarize recent advances in the DNA damage and replication checkpoints and discuss the essential functions of the proteins involved in the checkpoint responses.     

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.     

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.     

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: from initiation to recovery or adaptation

In response to diverse genotoxic stresses, cells activate DNA damage checkpoint pathways to protect genomic integrity and promote survival of the organism. Depending on DNA lesions and context, damaged cells with alarmed checkpoints can be eliminated by apoptosis or silenced by cellular senescence, or can survive and resume cell cycle progression upon checkpoint termination. Over the past two years a plethora of mechanistic studies have provided exciting insights into the biology and pathology of checkpoint initiation and signal propagation, and have revealed the various ways in which the response can be terminated: through recovery, adaptation or cancer-prone subversion. Such studies highlight the dynamic nature of these processes and help us to better understand the molecular basis, spatiotemporal orchestration and biological significance of the DNA damage response in normal and cancerous cells.     

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.     

DNA damage checkpoints inhibit mitotic exit by two different mechanisms

Cyclin-dependent kinase (CDK) governs cell cycle progression, and its kinase activity fluctuates during the cell cycle. Mitotic exit pathways are responsible for the inactivation of CDK after chromosome segregation by promoting the release of a nucleolus-sequestered phosphatase, Cdc14, which antagonizes CDK. In the budding yeast Saccharomyces cerevisiae, mitotic exit is controlled by the FEAR (for "Cdc-fourteen early anaphase release") and mitotic exit network (MEN) pathways. In response to DNA damage, two branches of the DNA damage checkpoint, Chk1 and Rad53, are activated in budding yeast to prevent anaphase entry and mitotic exit, allowing cells more time to repair damaged DNA. Here we present evidence indicating that yeast cells negatively regulate mitotic exit through two distinct pathways in response to DNA damage. Rad53 prevents mitotic exit by inhibiting the MEN pathway, whereas the Chk1 pathway prevents FEAR pathway-dependent Cdc14 release in the presence of DNA damage. In contrast to previous data, the Rad53 pathway negatively regulates MEN independently of Cdc5, a Polo-like kinase essential for mitotic exit. Instead, a defective Rad53 pathway alleviates the inhibition of MEN by Bfa1.     

Are there DNA damage checkpoints in E. coli?

The concept of regulatory ‘checkpoints’ in the eukaryotic cycle has proved to be a fruitful one. Here, its applicability to the bacterial cell cycle is examined. A primitive DNA damage checkpoint operates in E. coli such that, after exposure to ultraviolet light, while excision repair occurs, chromosome replication continues very slowly with the production of discontinuous daughter strands. The slower the rate of excision of photoproducts, the greater the delay before the normal rate of DNA replication is restored, the additional time for repair ensuring that normal survival is maintained. A model is proposed in which replication rate is controlled by the ratio of RecAcoated to uncoated single stranded regions of DNA in the replication fork. There are also two cell division inhibitors SulA (=SfiA) and SfiC under the control of the SOS system and sensitive to DNA damage, but they are irrelevant to the survival of wild-type bacteria under normal conditions. In strains where SulA and SfiC do not operate, inhibition is not influenced by the rate of excision repair and so fails one of the criteria for a DNA damage checkpoint, namely the monitoring of the DNA for the level of residual damage.     

Mammalian G1- and S-phase checkpoints in response to DNA damage

The ability to preserve genomic integrity is a fundamental feature of life. Recent findings regarding the molecular basis of the cell-cycle checkpoint responses of mammalian cells to genotoxic stress have converged into a two-wave concept of the G1 checkpoint, and shed light on the so-far elusive intra-S-phase checkpoint. Rapidly operating cascades that target the Cdc25A phosphatase appear central in both the initiation wave of the G1 checkpoint (preceding the p53-mediated maintenance wave) and the transient intra-S-phase response. Multiple links between defects in the G1/S checkpoints, genomic instability and oncogenesis are emerging, as are new challenges and hopes raised by this knowledge.     

Telomeres in drag: Dressing as DNA damage to engage telomerase

The telomere field concentrates both on mechanisms of telomere synthesis and the mechanisms by which telomeres protect chromosome termini from fusion and degradation. Recent studies show that the DNA damage response (DDR) machinery, formerly thought to be the culprit in deleterious telomeric fusion and degradation reactions, plays an active role not only in telomere protection but also in regulating telomere synthesis. Conversely, semi-conservative DNA replication, responsible for the bulk of telomere synthesis, now appears to be a pivotal event on the road to telomere de-protection. These advances prompt the notion that the two guises of telomere function are intricately entangled. Indeed, telomeres appear to expose themselves to the DDR upon passage of the replication fork, in turn attracting telomerase.     

DNA repair protein Rad55 is a terminal substrate of the DNA damage checkpoints

Checkpoints, which are integral to the cellular response to DNA damage, coordinate transient cell cycle arrest and the induced expression of DNA repair genes after genotoxic stress. DNA repair ensures cellular survival and genomic stability, utilizing a multipathway network. Here we report evidence that the two systems, DNA damage checkpoint control and DNA repair, are directly connected by demonstrating that the Rad55 double-strand break repair protein of the recombinational repair pathway is a terminal substrate of DNA damage and replication block checkpoints. Rad55p was specifically phosphorylated in response to DNA damage induced by the alkylating agent methyl methanesulfonate, dependent on an active DNA damage checkpoint. Rad55p modification was also observed after gamma ray and UV radiation. The rapid time course of phosphorylation and the recombination defects identified in checkpoint-deficient cells are consistent with a role of the DNA damage checkpoint in activating recombinational repair. Rad55p phosphorylation possibly affects the balance between different competing DNA repair pathways.     

ATM-dependent chromatin remodeler Rsf-1 facilitates DNA damage checkpoints and homologous recombination repair

As a member of imitation switch (ISWI) family in ATP-dependent chromatin remodeling factors, RSF complex consists of SNF2h ATPase and Rsf-1. Although it has been reported that SNF2h ATPase is recruited to DNA damage sites (DSBs) in a poly(ADP-ribosyl) polymerase 1 (PARP1)-dependent manner in DNA damage response (DDR), the function of Rsf-1 is still elusive. Here we show that Rsf-1 is recruited to DSBs confirmed by various cellular analyses. Moreover, the initial recruitment of Rsf-1 and SNF2h to DSBs shows faster kinetics than that of γH2AX after micro-irradiation. Signals of Rsf-1 and SNF2h are retained over 30 min after micro-irradiation, whereas γH2AX signals are gradually reduced at 10 min. In addition, Rsf-1 is accumulated at DSBs in ATM-dependent manner, and the putative pSQ motifs of Rsf-1 by ATM are required for its accumulation at DSBs. Furtheremore, depletion of Rsf-1 attenuates the activation of DNA damage checkpoint signals and cell survival upon DNA damage. Finally, we demonstrate that Rsf-1 promotes homologous recombination repair (HRR) by recruiting resection factors RPA32 and Rad51. Thus, these findings reveal a new function of chromatin remodeler Rsf-1 as a guard in DNA damage checkpoints and homologous recombination repair.     

Telomeres and the DNA damage response: why the fox is guarding the henhouse

DNA double strand breaks (DSBs) are repaired by an extensive network of proteins that recognize damaged DNA and catalyze its repair. By virtue of their similarity, the normal ends of linear chromosomes and internal DNA DSBs are both potential substrates for DSB repair enzymes. Thus, telomeres, specialized nucleo-protein complexes that cap chromosomal ends, serve a critical function to differentiate themselves from internal DNA strand breaks, and as a result prevent genomic instability that can result from their inappropriate involvement in repair reactions. Telomeres that become critically short due to failure of telomere maintenance mechanisms, or which become dysfunctional by loss of telomere binding proteins, elicit extensive checkpoint responses that in normal cells blocks proliferation. In this situation, the DNA DSB repair machinery plays a major role in responding to these "damaged" telomeres - creating chromosome fusions or capturing telomeres from other chromosomes in an effort to rid the cell of the perceived damage. However, a surprising aspect of telomere maintenance is that many of the same proteins that facilitate this repair of damaged telomeres are also necessary for their proper integrity. Here, we review recent work defining the roles for DSB repair machinery in telomere maintenance and in response to telomere dysfunction.