The histone methyltransferase Dot1 is required for DNA damage repair and proper development in Dictyostelium

Posttranslational histone modifications play an important role in modulating gene expression and chromatin structure. Here we report the identification of histone H3K79 dimethylation in the simple eukaryote Dictyostelium discoideum. We have deleted the D. discoideum Dot1/KMT4 homologue and demonstrate that it is the sole enzyme responsible for histone H3K79me2. Cells lacking Dot1 are reduced in growth and delayed in development, but do not show apparent changes in cell cycle regulation. Furthermore, our results indicate that Dot1 contributes to UV damage resistance and DNA repair in D. discoideum. In summary, the data support the view that the machinery controlling the setting of histone marks is evolutionary highly conserved and provide evidence that D. discoideum is a suitable model system to analyze these modifications and their functions during development and differentiation.     

Drosophila melanogaster: a model for the study of DNA damage checkpoint response

The cells of metazoans respond to DNA damage by either arresting their cell cycle in order to repair the DNA, or by undergoing apoptosis. This response is highly conserved across species, and many of the genes involved in this DNA damage response have been shown to be inactivated in human cancers. This suggests the importance of DNA damage response with regard to the prevention of cancer. The DNA damage checkpoint responses vary greatly depending on the developmental context, cell type, gene expression profile, and the degree and nature of the DNA lesions. More valuable information can be obtained from studies utilizing whole organisms in which the molecular basis of development has been well established, such as Drosophila. Since the discovery of the Drosophila p53 orthologue, various aspects of DNA damage responses have been studied in Drosophila. In this review, I will summarize the current knowledge on the DNA damage checkpoint response in Drosophila. With the ease of genetic, cellular, and cytological approaches, Drosophila will become an increasingly valuable model organism for the study of mechanisms inherent to cancer formation associated with defects in the DNA damage pathway.     

Surveillance mechanisms monitoring chromosome breaks during mitosis and meiosis

DNA double-strand breaks (DSBs) are highly hazardous for genome integrity, because failure to repair them can lead to genome rearrangements or chromosome loss. They can arise at unpredictable locations as a consequence of DNA damage during both the mitotic and the meiotic cell cycle or in a programmed manner during meiosis. Cellular response to accidental or programmed DSBs involves highly conserved surveillance mechanisms, called DNA damage checkpoint and recombination checkpoint, which coordinate DSB repair with mitotic or meiotic cell cycle progression, respectively. Although these protective signal-transduction pathways share several upstream components, activation of the recombination checkpoint requires meiosis-specific proteins. These proteins are structural components of the meiotic chromosomes, indicating that the system monitoring programmed meiotic DSBs is an integral part of the chromosome structure formed during meiosis.     

Human Pluripotent Stem Cells Have a Novel Mismatch Repair-dependent Damage Response

Human pluripotent stem cells (PSCs) are presumed to have robust DNA repair pathways to ensure genome stability. PSCs likely need to protect against mutations that would otherwise be propagated throughout all tissues of the developing embryo. How these cells respond to genotoxic stress has only recently begun to be investigated. Although PSCs appear to respond to certain forms of damage more efficiently than somatic cells, some DNA damage response pathways such as the replication stress response may be lacking. Not all DNA repair pathways, including the DNA mismatch repair (MMR) pathway, have been well characterized in PSCs to date. MMR maintains genomic stability by repairing DNA polymerase errors. MMR is also involved in the induction of cell cycle arrest and apoptosis in response to certain exogenous DNA-damaging agents. Here, we examined MMR function in PSCs. We have demonstrated that PSCs contain a robust MMR pathway and are highly sensitive to DNA alkylation damage in an MMR-dependent manner. Interestingly, the nature of this alkylation response differs from that previously reported in somatic cell types. In somatic cells, a permanent G₂/M cell cycle arrest is induced in the second cell cycle after DNA damage. The PSCs, however, directly undergo apoptosis in the first cell cycle. This response reveals that PSCs rely on apoptotic cell death as an important defense to avoid mutation accumulation. Our results also suggest an alternative molecular mechanism by which the MMR pathway can induce a response to DNA damage that may have implications for tumorigenesis.     

Viral manipulation of DNA repair and cell cycle checkpoints

Recognition and repair of DNA damage is critical for maintaining genomic integrity and suppressing tumorigenesis. In eukaryotic cells, the sensing and repair of DNA damage are coordinated with cell cycle progression and checkpoints, in order to prevent the propagation of damaged DNA. The carefully maintained cellular response to DNA damage is challenged by viruses, which produce a large amount of exogenous DNA during infection. Viruses also express proteins that perturb cellular DNA repair and cell cycle pathways, promoting tumorigenesis in their quest for cellular domination. This review presents an overview of strategies employed by viruses to manipulate DNA damage responses and cell cycle checkpoints as they commandeer the cell to maximize their own viral replication. Studies of viruses have identified key cellular regulators and revealed insights into molecular mechanisms governing DNA repair, cell cycle checkpoints, and transformation.     

Induction of p53-, MDM2-, and WAF1/CIP1-like Molecules in Insect Cells by DNA-Damaging Agents

Cellular responses following DNA damage are ubiquitous in the biological world. In response to DNA damage, cell cycle checkpoints are activated, which delay cell cycle progression and most likely serve to allow time for repair. One important checkpoint in mammalian cells, activated in the G₁ phase of the cell cycle, is dependent on the p53 tumor suppressor gene product. While p53 is responsible for inducing G₁ arrest, the product of the MDM2 gene is believed to alleviate the arrest, allowing continuation of the cell cycle after a transient delay. Inasmuch as MDM2 and WAF1/CIP1 are transactivated by p53, while MDM2 binds to and modulates the activity of p53, a "feedback loop" is thus created. This pathway has been highly conserved in mammalian cells, but its presence outside of vertebrates is unknown. By using human MDM2 and WAF1/CIP1 cDNA probes, and monoclonal antibodies to p53 and Mdm2, we demonstrate in insect cell lines evidence for the existence of p53-, MDM2-, and WAF1/CIP1 -like molecules and a p53-regulated pathway following treatment by DNA-damaging agents.     

Studying the DNA damage response using in vitro model systems

Exogenous and endogenous insults continuously damage DNA. DNA damage must be detected in order to prevent loss of vital genetic information. Cells respond to DNA damage by activating checkpoint pathways that delay the progression through the cell cycle, promote DNA repair or induce cell death. A regulatory network of proteins has been identified that participate in DNA damage checkpoint pathways. Central to this network are ATM, ATR and the Mre11/Rad50/Nbs1 (MRN) complex. Detailed biochemical analysis of ATM, ATR and the MRN dependent DNA damage responses has taken advantage of several in vitro model systems to understand the detailed mechanisms underlying their function. Here we describe some recent findings obtained analysing these pathways using in vitro model systems. In particular we focus on the studies performed in the Xenopus laevis egg cell free extract, which recapitulates the DNA damage response in the context of the cell cycle.     

A Mitotic Phosphorylation Feedback Network Connects Cdk1, Plk1, 53BP1, and Chk2 to Inactivate the G2/M DNA Damage Checkpoint

DNA damage checkpoints arrest cell cycle progression to facilitate DNA repair. The ability to survive genotoxic insults depends not only on the initiation of cell cycle checkpoints but also on checkpoint maintenance. While activation of DNA damage checkpoints has been studied extensively, molecular mechanisms involved in sustaining and ultimately inactivating cell cycle checkpoints are largely unknown. Here, we explored feedback mechanisms that control the maintenance and termination of checkpoint function by computationally identifying an evolutionary conserved mitotic phosphorylation network within the DNA damage response. We demonstrate that the non-enzymatic checkpoint adaptor protein 53BP1 is an in vivo target of the cell cycle kinases Cyclin-dependent kinase-1 and Polo-like kinase-1 (Plk1). We show that Plk1 binds 53BP1 during mitosis and that this interaction is required for proper inactivation of the DNA damage checkpoint. 53BP1 mutants that are unable to bind Plk1 fail to restart the cell cycle after ionizing radiation-mediated cell cycle arrest. Importantly, we show that Plk1 also phosphorylates the 53BP1-binding checkpoint kinase Chk2 to inactivate its FHA domain and inhibit its kinase activity in mammalian cells. Thus, a mitotic kinase-mediated negative feedback loop regulates the ATM-Chk2 branch of the DNA damage signaling network by phosphorylating conserved sites in 53BP1 and Chk2 to inactivate checkpoint signaling and control checkpoint duration.     

Bacterial endospores and their significance in stress resistance

In terms of resistance to extreme environmental stresses, the bacterial spore represents a pinnacle of evolution. Spores are highly resistant to a wide variety of physical stresses such as: wet and dry heat, UV and gamma radiation, oxidizing agents, chemicals, and extremes of both vacuum and ultrahigh hydrostatic pressure. Some of the molecular mechanisms underlying spore resistance properties have been elucidated in the laboratory, and involve both: (i) protection of vital spore macromolecules during dormancy, and (ii) repair of damaged macromolecules during germination. Our group has recently become interested in testing if the laboratory model of spore UV resistance is relevant to spore persistence in the environment. We have constructed a number of Bacillus subtilis strains which are defective in various DNA repair systems and spore structural components. Using spores of these strains, we have been exploring: (i) the types of damage induced in DNA by the UV-B and UV-A components of sunlight; (ii) the relative contribution of the major spore DNA repair systems to spore solar radiation resistance; and (iii) the role of spore structural components such as the spore coats and dipicolinic acid (DPA) in attenuation of the lethal and mutagenic effects of solar UV. The current data are reviewed with the ultimate goal of obtaining a complete model describing spore persistence and longevity in the terrestrial solar UV radiation environment. This revised version was published online in August 2006 with corrections to the Cover Date.     

Fructokinase is required for carbon partitioning to cellulose in aspen wood

DNA damage checkpoints delay mitotic cell‐cycle progression in response to DNA stress, stalling the cell cycle to allow time for repair. CDKB is a plant‐specific cyclin‐dependent kinase (CDK) that is required for the G₂/M transition of the cell cycle. In Arabidopsis, DNA damage leads the degradation of CDKB2, and the subsequent G₂ arrest gives cells time to repair damaged DNA. G₂ arrest also triggers transition from the mitotic cycle to endoreduplication, leading to the presence of polyploid cells in many tissues. In contrast, in rice (Oryza sativa), polyploid cells are found only in the endosperm. It was unclear whether endoreduplication contributes to alleviating DNA damage in rice (Oryza sativa). Here, we show that DNA damage neither down‐regulates Orysa;CDKB2;1 nor induces endoreduplication in rice. Furthermore, we found increased levels of Orysa;CDKB2;1 protein upon DNA damage. These results suggest that CDKB2 functions differently in Arabidopsis and rice in response to DNA damage. Arabidopsis may adopt endoreduplication as a survival strategy under genotoxic stress conditions, but rice may enhance DNA repair capacity upon genotoxic stress. In addition, polyploid cells due to endomitosis were present in CDKB2;1 knockdown rice, suggesting an important role for Orysa;CDKB2;1 during mitosis.     

A plant solution to the CDK conundrum in the DNA damage response

To cope with DNA damage, proliferating cells have evolved sophisticated mechanisms including cell cycle arrest and activation of DNA repair. Paradoxically, various DNA damage response pathways are promoted by cyclin‐dependent kinase (CDK) activity, while cell cycle remains arrested. New work in The EMBO Journal shows that plant cells have evolved intricate ways to resolve this dilemma, by utilizing distinct and specialized CDKs for cell cycle progression and homologous recombination.     

The nucleotide excision repair pathway is required for UV-C-induced apoptosis in Caenorhabditis elegans

Ultraviolet (UV) radiation is a mutagen of major clinical importance in humans. UV-induced damage activates multiple signaling pathways, which initiate DNA repair, cell cycle arrest and apoptosis. To better understand these pathways, we studied the responses to UV-C light (254 nm) of germ cells in Caenorhabditis elegans. We found that UV activates the same cellular responses in worms as in mammalian cells. Both UV-induced apoptosis and cell cycle arrest were completely dependent on the p53 homolog CEP-1, the checkpoint proteins HUS-1 and CLK-2, and the checkpoint kinases CHK-2 and ATL-1 (the C. elegans homolog of ataxia telangiectasia and Rad3-related); ATM-1 (ataxia telangiectasia mutated-1) was also required, but only at low irradiation doses. Importantly, mutation of genes encoding nucleotide excision repair pathway components severely disrupted both apoptosis and cell cycle arrest, suggesting that these genes not only participate in repair, but also signal the presence of damage to downstream components of the UV response pathway that we delineate here. Our study suggests that whereas DNA damage response pathways are conserved in metazoans in their general outline, there is significant evolution in the relative importance of individual checkpoint genes in the response to specific types of DNA damage.     

Regulation of the DNA Damage Response by Cyclin-Dependent Kinases

The eukaryotic cell cycle comprises a series of events, whose ordering and correct progression depends on the oscillating activity of cyclin-dependent kinases (Cdks), which safeguard timely duplication and segregation of the genome. Cell division is intimately connected to an evolutionarily conserved DNA damage response (DDR), which involves DNA repair pathways that reverse DNA lesions, as well as checkpoint pathways that inhibit cell cycle progression while repair occurs. There is increasing evidence that Cdks are involved in the DDR, in particular in DNA repair by homologous recombination and in activation of the checkpoint response. However, Cdks have to be carefully regulated, because even an excess of their activity can affect genome stability. In this review, we consider the physiological role of Cdks in the DDR.