Targeted deletion of mouse Rad1 leads to deficient cellular DNA damage responses

The Rad1 gene is evolutionarily conserved from yeast to human. The fission yeast Schizosaccharomyces pombeRad1 ortholog promotes cell survival against DNA damage and is required for G₂/M checkpoint activation. In this study, mouse embryonic stem (ES) cells with a targeted deletion of Mrad1, the mouse ortholog of this gene, were created to evaluate its function in mammalian cells. Mrad1^-/- ES cells were highly sensitive to ultraviolet-light (UV light), hydroxyurea (HU) and gamma rays, and were defective in G₂/M as well as S/M checkpoints. These data indicated that Mrad1 is required for repairing DNA lesions induced by UV-light, HU and gamma rays, and for mediating G₂/M and S/M checkpoint controls. We further demonstrated that Mrad1 plays an important role in homologous recombination repair (HRR) in ES cells, but a minor HRR role in differentiated mouse cells.     

The novel DNA damage checkpoint protein ddc1p is phosphorylated periodically during the cell cycle and in response to DNA damage in budding yeast.

The DDC1 gene was identified, together with MEC3 and other checkpoint genes, during a screening for mutations causing synthetic lethality when combined with a conditional allele altering DNA primase. Deletion of DDC1 causes sensitivity to UV radiation, methyl methanesulfonate (MMS) and hydroxyurea (HU). ddc1Delta mutants are defective in delaying G1-S and G2-M transition and in slowing down the rate of DNA synthesis when DNA is damaged during G1, G2 or S phase, respectively. Therefore, DDC1 is involved in all the known DNA damage checkpoints. Conversely, Ddc1p is not required for delaying entry into mitosis when DNA synthesis is inhibited. ddc1 and mec3 mutants belong to the same epistasis group, and DDC1 overexpression can partially suppress MMS and HU sensitivity of mec3Delta strains, as well as their checkpoint defects. Moreover, Ddc1p is phosphorylated periodically during a normal cell cycle and becomes hyperphosphorylated in response to DNA damage. Both phosphorylation events are at least partially dependent on a functional MEC3 gene.     

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.     

DNA damage responses to oxidative stress

The DNA damage response is a hierarchical process. DNA damage is detected by sensor proteins such as the MRN complex that transmit the information to transducer proteins such as ATM and ATR, which control the damage response through the phosphorylation of effector proteins. The extent of the DNA damage determines cell fate: cell cycle arrest and DNA repair or the activation of apoptotic pathways. In aerobic cells, reactive oxygen species (ROS) are generated as a by-product of normal mitochondrial activity. If not properly controlled, ROS can cause severe damage to cellular macromolecules, especially the DNA. We describe here some of the cellular responses to alterations in the cellular redox state during hypoxia or oxidative stress. Oxidative damage in DNA is repaired primarily via the base excision repair (BER) pathway which appears to be the simplest of the three excision repair pathways. To allow time for DNA repair, the cells activate their cell cycle checkpoints, leading to cell cycle arrest and preventing the replication of damage and defective DNA.     

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 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.     

Functional interaction between FOXO3a and ATM regulates DNA damage response

The maintenance of genomic stability in cells is relentlessly challenged by environmental stresses that induce DNA breaks, which activate the DNA-damage pathway mediated by ataxia-telangiectasia mutated (ATM) and its downstream mediators to control damage-induced cell-cycle checkpoints and DNA repair. Here, we show that FOXO3a interacts with ATM to promote phosphorylation of ATM at Ser 1981 and prompting its downstream mediators to form nuclear foci in response to DNA damage. Silencing FOXO3a in cells abrogates the formation of ATM-pS1981 and phospho-histone H2AX foci after DNA damage. Increasing FOXO3a in cells promotes ATM-regulated signalling, the intra-S-phase or G2-M cell-cycle checkpoints, and the repair of damaged DNA, whereas cells lacking FOXO3a did not trigger the DNA-repair mechanism after DNA damage. The carboxy-terminal domain of FOXO3a binds to the FAT domain of ATM, thereby contributing to the activation of ATM. These results suggest that ATM may be regulated directly by FOXO3a in the DNA-damage response.     

Low efficiency of repair of critical DNA damage induced by low doses of radiation

This study provides an analysis of the development of cellular response to the critical DNA damage and the mechanisms for limiting the efficiency of repairing such damages induced by low doses of ionizing radiation exposure. Based on the data of many studies, one can conclude that the majority of damages occurring in the DNA of the cells after exposure to ionizing radiation significantly differ in their chemical nature from the endogenous ones. The most important characteristic of radiation-induced DNA damages is their complexity and clustering. Double strand breaks, interstrand crosslinks or destruction of the replication fork and formation of long single-stranded gaps in DNA are considered to be critical damages for the fate of cells. The occurrence of such lesions in DNA may be a key event in the etiology and the therapy of cancer. The appearance in the cells of the critical DNA damage induces a rapid development of a complex and ramified network of molecular and biochemical reactions which are called the cellular response to DNA damage. Induction of the cellular response to DNA damage involves the activation of the systems of cell cycle checkpoints, DNA repair, changes in the expression of many genes, reconstruction of the chromatin or apoptosis. However, the efficiency of repair of the complex DNA damage in cells after exposure to low doses of radiation remains at low levels. The development of the cell response to DNA damages after exposure to low doses of radiation does not reach the desired result due to a small amount of damage, with the progression of the phase cell cycle being ahead of the processes of DNA repair. This is primarily due to the failure of signalization to activate the checkpoint of the cell cycle for its arrest in the case of a small number of critical DNA lesions. In the absence of the arrest of the phase cell cycle progression, especially during the G2/M transition, the reparation mechanisms fail to completely restore DNA, and cells pass into mitosis with a damaged DNA. It is assumed that another reason for the low efficiency of DNA repair in the cells after exposure to low doses of radiation is the existence of a restricted access for the repair system components to the complex damages at the DNA sites of highly compacted chromatin.     

Human CST abundance determines recovery from diverse forms of DNA damage and replication stress

Mammalian CST (CTC1-STN1-TEN1) is a telomere-associated complex that functions in telomere duplex replication and fill-in synthesis of the telomeric C-strand following telomerase action. CST also facilitates genome-wide replication recovery after HU-induced fork stalling by increasing origin firing. CTC1 and STN1 were originally isolated as a DNA polymerase α stimulatory factor. Here we explore how CST abundance affects recovery from drugs that cause different types of DNA damage and replication stress. We show that recovery from HU and aphidicolin induced replication stress is increased by CST over-expression. Elevated CST increases dNTP incorporation and origin firing after HU release and decreases the incidence of anaphase bridges and micronuclei after aphidicolin removal. While the frequency of origin firing after HU release is proportional to CST abundance, the number of cells entering S-phase to initiate replication is unchanged by CST overexpression or STN1 depletion. Instead the CST-related changes in origin firing take place in cells that were already in S-phase at the time of HU addition, indicating that CST modulates firing of late or dormant origins. CST abundance also influences cell viability after treatment with HU, aphidicolin, MMS and camptothecin. Viability is increased by elevated CST and decreased by STN1 depletion, indicating that endogenous CST levels are limiting. However, CST abundance does not affect viability after MMC treatment. Thus, CST facilitates recovery from many, but not all, forms of exogenous DNA damage. Overall our results suggest that CST is needed in stoichiometric amounts to facilitate re-initiation of DNA replication at repaired forks and/or dormant origins.     

Persistent DNA damage signaling and DNA polymerase theta promote broken chromosome segregation

Cycling cells must respond to DNA double-strand breaks (DSBs) to avoid genome instability. Missegregation of chromosomes with DSBs during mitosis results in micronuclei, aberrant structures linked to disease. How cells respond to DSBs during mitosis is incompletely understood. We previously showed that Drosophila melanogaster papillar cells lack DSB checkpoints (as observed in many cancer cells). Here, we show that papillar cells still recruit early acting repair machinery (Mre11 and RPA3) and the Fanconi anemia (FA) protein Fancd2 to DSBs. These proteins persist as foci on DSBs as cells enter mitosis. Repair foci are resolved in a stepwise manner during mitosis. DSB repair kinetics depends on both monoubiquitination of Fancd2 and the alternative end-joining protein DNA polymerase θ. Disruption of either or both of these factors causes micronuclei after DNA damage, which disrupts intestinal organogenesis. This study reveals a mechanism for how cells with inactive DSB checkpoints can respond to DNA damage that persists into mitosis.     

DNA damage in blood leukocytes of individuals with sickle cell disease treated with hydroxyurea

Hydroxyurea (HU) plays an important role in the treatment of patients with sickle cell disease (SCD). Although HU has been associated with an increased risk of leukemia in some patients with myeloproliferative disorders, the mutagenic and carcinogenic potential of HU has not been established. This study investigated levels of DNA damage using the alkaline (pH>13) comet assay to analyze peripheral blood leukocytes sampled from 28 patients with SCD treated with HU (SCHU) and from 28 normal individuals. The damage index (DI) in the SCHU group was significantly higher than in controls (p<0.05). Gender, smoking or age were not associated with DNA damage in controls or SCHU individuals. In the group of SCHU individuals, mean HU dose and DI were positively correlated, and individuals who received a mean dose of >20 mg/kg HU (DI=24.9+/-5.5) showed significantly more DNA damage than those who received < or =20 mg/kg HU (DI=14.6+/-1.8) (p<0.05). Individuals treated for > or =42 months (DI=23.1+/-4.2) showed significantly greater DNA damage than those treated for <42 months (13.6+/-1.9) (p<0.05). DI was inversely correlated with body mass index in the SCHU group.     

The DNA damage checkpoint pathways exert multiple controls on the efficiency and outcome of the repair of a double-stranded DNA gap

A DNA gap repair assay was used to determine the effect of mutations in the DNA damage checkpoint system on the efficiency and outcome (crossover/non-crossover) of recombinational DNA repair. In Saccharomyces cerevisiae gap repair is largely achieved by homologous recombination. As a result the plasmid either integrates into the chromosome (indicative of a crossover outcome) or remains extrachromosomal (indicative of a non-crossover outcome). Deletion mutants of the MEC1 and RAD53 checkpoint kinase genes exhibited a 5-fold decrease in gap repair efficiency, showing that 80% of the gap repair events depended on functional DNA damage checkpoints. Epistasis analysis suggests that the DNA damage checkpoints affect gap repair by modulating Rad51 protein-mediated homologous recombination. While in wild-type cells only ~25% of the gap repair events were associated with a crossover outcome, Mec1-deficient cells exhibited a >80% crossover association. Also mutations in the effector kinases Rad53, Chk1 and Dun1 were found to affect crossover association of DNA gap repair to various degrees. The data suggest that the DNA damage checkpoints are important for the optimal functioning of recombinational DNA repair with multiple terminal targets to modulate the efficiency and outcome of homologous recombination.     

Decreased MCM2-6 in Drosophila S2 cells does not generate significant DNA damage or cause a marked increase in sensitivity to replication interference

A reduction in the level of some MCM proteins in human cancer cells (MCM5 in U20S cells or MCM3 in Hela cells) causes a rapid increase in the level of DNA damage under normal conditions of cell proliferation and a loss of viability when the cells are subjected to replication interference. Here we show that Drosophila S2 cells do not appear to show the same degree of sensitivity to MCM2-6 reduction. Under normal cell growth conditions a reduction of >95% in the levels of MCM3, 5, and 6 causes no significant short term alteration in the parameters of DNA replication or increase in DNA damage. MCM depleted cells challenged with HU do show a decrease in the density of replication forks compared to cells with normal levels of MCM proteins, but this produces no consistent change in the levels of DNA damage observed. In contrast a comparable reduction of MCM7 levels has marked effects on viability, replication parameters and DNA damage in the absence of HU treatment.     

Inhibition of ATR protein kinase activity by schisandrin B in DNA damage response

ATM and ATR protein kinases play a crucial role in cellular DNA damage responses. The inhibition of ATM and ATR can lead to the abolition of the function of cell cycle checkpoints. In this regard, it is expected that checkpoint inhibitors can serve as sensitizing agents for anti-cancer chemo/radiotherapy. Although several ATM inhibitors have been reported, there are no ATR-specific inhibitors currently available. Here, we report the inhibitory effect of schisandrin B (SchB), an active ingredient of Fructus schisandrae, on ATR activity in DNA damage response. SchB treatment significantly decreased the viability of A549 adenocarcinoma cells after UV exposure. Importantly, SchB treatment inhibited both the phosphorylation levels of ATM and ATR substrates, as well as the activity of the G2/M checkpoint in UV-exposed cells. The protein kinase activity of immunoaffinity-purified ATR was dose-dependently decreased by SchB in vitro (IC₅₀: 7.25 μM), but the inhibitory effect was not observed in ATM, Chk1, PI3K, DNA-PK, and mTOR. The extent of UV-induced phosphorylation of p53 and Chk1 was markedly reduced by SchB in ATM-deficient but not siATR-treated cells. Taken together, our demonstration of the ability of SchB to inhibit ATR protein kinase activity following DNA damage in cells has clinical implications in anti-cancer therapy.     

The role of inhibitory phosphorylation of CDC2 following DNA replication block and radiation-induced damage in human cells.

It has been suggested that the survival response of p53 defective tumor cells to agents that inhibit DNA replication or damage DNA may be largely dependent on cell cycle checkpoints that regulate the onset of mitosis. In human cells, the mitosis-inducing kinase CDC2/cyclin B is inhibited by phosphorylation of threonine-14 and tyrosine-15, but the roles of these phosphorylations in enforcing checkpoints is not known. We have investigated the situation in a human cervical carcinoma cell line (HeLa cells) and found that low level expression of a mutant nonphosphorylatable form of CDC2 abrogates regulation of the endogenous CDC2/cyclin B. Disruption of this pathway is toxic and renders cells highly sensitive to killing by DNA damage or by inhibition of DNA replication. These findings establish the importance of inhibitory phosphorylation of CDC2 in the survival mechanism used by human cells when exposed to some of the most common forms of anticancer therapy.