Use of algae in the study of essential cell processes

Maintenance of genomic stability is of crucial importance for all living organisms. It is no surprise that during evolution, a series of highly selective and efficient systems to detect DNA damage and control its repair have evolved. To this end, signal transduction pathways are involved in pausing the cell division cycle to provide time for repair, and ultimately releasing the cell cycle from arrest. Genetic components of the damage and replication checkpoints have been identified and a working model is beginning to emerge. This area of biological inquiry has received a great deal of attention in the past decade with the realization that the underlying regulatory mechanisms controlling the cell cycle are conserved throughout eukaryotic evolution. Many of the key players in this response have structural and functional counterparts in species as diverse as yeast and human. In recent years attention has also been paid to the plant kingdom suggesting that checkpoint controls have been highly conserved during evolution. The unicellular green alga Chlamydomonas reinhardtii is a suitable model organism for the study of basic cellular processes including cell cycle regulation and DNA repair. To investigate how algal cells accomplish these tasks, we have isolated mutants in the recognition and repair of DNA damage or in the response to DNA damage. Presented at the International Symposium Biology and Taxonomy of Green Algae V, Smolenice, June 26–29, 2007, Slovakia.     

A molecular bar-coded DNA repair resource for pooled toxicogenomic screens

DNA damage from exogenous and endogenous sources can promote mutations and cell death. Fortunately, cells contain DNA repair and damage signaling pathways to reduce the mutagenic and cytotoxic effects of DNA damage. The identification of specific DNA repair proteins and the coordination of DNA repair pathways after damage has been a central theme to the field of genetic toxicology and we have developed a tool for use in this area. We have produced 99 molecular bar-coded Escherichia coli gene-deletion mutants specific to DNA repair and damage signaling pathways, and each bar-coded mutant can be tracked in pooled format using bar-code specific microarrays. Our design adapted bar-codes developed for the Saccharomyces cerevisiae gene-deletion project, which allowed us to utilize an available microarray product for pooled gene-exposure studies. Microarray-based screens were used for en masse identification of individual mutants sensitive to methyl methanesulfonate (MMS). As expected, gene-deletion mutants specific to direct, base excision, and recombinational DNA repair pathways were identified as MMS-sensitive in our pooled assay, thus validating our resource. We have demonstrated that molecular bar-codes designed for S. cerevisiae are transferable to E. coli, and that they can be used with pre-existing microarrays to perform competitive growth experiments. Further, when comparing microarray to traditional plate-based screens both overlapping and distinct results were obtained, which is a novel technical finding, with discrepancies between the two approaches explained by differences in output measurements (DNA content versus cell mass). The microarray-based classification of Deltatag and DeltadinG cells as depleted after MMS exposure, contrary to plate-based methods, led to the discovery that Deltatag and DeltadinG cells show a filamentation phenotype after MMS exposure, thus accounting for the discrepancy. A novel biological finding is the observation that while DeltadinG cells filament in response to MMS they exhibit wild-type sulA expression after exposure. This decoupling of filamentation from SulA levels suggests that DinG is associated with the SulA-independent filamentation pathway.     

Characterization of Postreplication Repair in Mutagen-Sensitive Strains of DROSOPHILA MELANOGASTER

Mutants of Drosophila melanogaster, with suspected repair deficiencies, were analyzed for their capacity to repair damage induced by X-rays and UV radiation. Analysis was performed on cell cultures derived from embryos of homozygous mutant stocks. Postreplication repair following UV radiation has been analyzed in mutant stocks derived from a total of ten complementation groups. Cultures were irradiated, pulse-labeled, and incubated in the dark prior to analysis by alkaline sucrose gradient centrifugation. Kinetics of the molecular weight increase in newly synthesized DNA were assayed after cells had been incubated in the presence or absence of caffeine. Two separate pathways of postreplication repair have been tentatively identified by mutants derived from four complementation groups. The proposed caffeine sensitive pathway (CAS) is defined by mutants which also disrupt meiosis. The second pathway (CIS) is caffeine insensitive and is not yet associated with meiotic functions. All mutants deficient in postreplication repair are also sensitive to nitrogen mustard. The mutants investigated display a normal capacity to repair single-strand breaks induced in DNA by X-rays, although two may possess a reduced capacity to repair damage caused by localized incorporation of high specific activity thymidine-³H. The data have been employed to construct a model for repair of UV-induced damage in Drosophila DNA. Implications of the model for DNA repair in mammals are discussed.     

Repair of UV damage in the fission yeast Schizosaccharomyces pombe

This review is concerned with repair and tolerance of UV damage in the fission yeast, Schizosaccharomyces pombe and with the differences between Sch. pombe and budding yeast, Saccharomyces cerevisiae in their response to UV irradiation. Sch. pombe is not as sensitive to ultra-violet radiation as Sac. cerevisiae nor are any of its mutants as sensitive as the most sensitive Sac. cerevisiae mutants. This can be explained in part by the fact that Sch. pombe, unlike budding yeast or mammalian cells, has an extra pathway (UVER) for excision of UV photoproducts in addition to nucleotide excision repair (NER). However, even in mutants lacking this additional pathway, there are significant differences between the two yeasts. Sch. pombe mutants that lack the alternative pathway are still more UV-resistant than wild-type Sac. cerevisiae; recombination mutants are significantly UV sensitive (unlike their Sac. cerevisiae equivalents); mutants lacking the second pathway are sensitized to UV by caffeine; and checkpoint mutants are relatively more sensitive than the budding yeast equivalents. In addition, Sch. pombe has no photolyase. Thus, the response to UV in the two yeasts has a number of significant differences, which are not accounted for entirely by the existence of two alternative excision repair pathways. The long G2 in Sch. pombe, its well-developed recombination pathways and efficient cell cycle checkpoints are all significant components in survival of UV damage.     

The cellular responses to DNA damage

The ability to survive spontaneous and induced DNA damage, and to minimize the number of heritable mutations that this causes, is essential to the maintenance of genome integrity for all organisms. Early studies on model eukaryotes focused on genes acting in defined DNA repair pathways. More recent work with the budding and fission yeasts and mammalian cells has started to integrate the DNA damage response with cell physiology and the cell cycle.     

The Aspergillus nidulans uvsB gene encodes an ATM-related kinase required for multiple facets of the DNA damage response

In Aspergillus nidulans, uvsB and uvsD belong to the same epistasis group of DNA repair mutants. Recent observations suggest that these genes are likely to control cell cycle checkpoint responses to DNA damage and incomplete replication. Consistent with this notion, we show here that UVSB is a member of the conserved family of ATM-related kinases. Phenotypic characterization of uvsB mutants shows that they possess defects in additional aspects of the DNA damage response besides checkpoint control, including inhibition of septum formation, regulation of gene expression, and induced mutagenesis. The musN227 mutation partially suppresses the poor growth and DNA damage sensitivity of uvsB mutants. Although musN227 partially suppresses several uvsB defects, it does not restore checkpoint function to uvsB mutants. Notably, the failure of uvsB mutants to restrain septum formation in the presence of DNA damage is suppressed by the musN227 mutation. We propose that UVSB functions as the central regulator of the A. nidulans DNA damage response, whereas MUSN promotes recovery by modulating a subset of the response.     

Multiple pathways for repair of hydrogen peroxide-induced DNA damage in Escherichia coli.

The repair response of Escherichia coli to hydrogen peroxide has been examined in mutants which show increased sensitivity to this agent. Four mutants were found to show increased in vivo sensitivity to hydrogen peroxide compared with wild type. These mutants, in order of increasing sensitivity, were recA, polC, xthA, and polA. The polA mutants were the most sensitive, implying that DNA polymerase I is required for any repair of hydrogen peroxide damage. Measurement of repair synthesis after hydrogen peroxide treatment demonstrated normal levels for recA mutants, a small amount for xthA mutants, and none for polA mutants. This is consistent with exonuclease III being required for part of the repair synthesis seen, while DNA polymerase I is strictly required for all repair synthesis. Sedimentation analysis of cellular DNA after hydrogen peroxide treatment showed that reformation was absent in xthA, polA, and polC(Ts) strains but normal in a recA cell line. By use of a lambda phage carrying a recA-lacZ fusion, we found hydrogen peroxide does not induce the recA promoter. Our findings indicate two pathways of repair for hydrogen peroxide-induced DNA damage. One of these pathways would utilize exonuclease III, DNA polymerase III, and DNA polymerase I, while the other would be DNA polymerase I dependent. The RecA protein seems to have little or no direct function in either repair pathway.     

MEC3, MEC1, and DDC2 are essential components of a telomere checkpoint pathway required for cell cycle arrest during senescence in Saccharomyces cerevisiae

When telomerase is absent and/or telomeres become critically short, cells undergo a progressive decline in viability termed senescence. The telomere checkpoint model predicts that cells will respond to a damaged or critically short telomere by transiently arresting and activating repair of the telomere. We examined the senescence of telomerase-deficient Saccharomyces cerevisiae at the cellular level to ask if the loss of telomerase activity triggers a checkpoint response. As telomerase-deficient mutants were serially subcultured, cells exhibited a progressive decline in average growth rate and an increase in the number of cells delayed in the G2/M stage of the cell cycle. MEC3, MEC1, and DDC2, genes important for the DNA damage checkpoint response, were required for the cell cycle delay in telomerase-deficient cells. In contrast, TEL1, RAD9, and RAD53, genes also required for the DNA damage checkpoint response, were not required for the G2/M delay in telomerase-deficient cells. We propose that the telomere checkpoint is distinct from the DNA damage checkpoint and requires a specific set of gene products to delay the cell cycle and presumably to activate telomerase and/or other telomere repair activities.     

Isolation of SOS constitutive mutants of Escherichia coli

The bacterial SOS regulon is strongly induced in response to DNA damage from exogenous agents such as UV radiation and nalidixic acid. However, certain mutants with defects in DNA replication, recombination, or repair exhibit a partially constitutive SOS response. These mutants presumably suffer frequent replication fork failure, or perhaps they have difficulty rescuing forks that failed due to endogenous sources of DNA damage. In an effort to understand more clearly the endogenous sources of DNA damage and the nature of replication fork failure and rescue, we undertook a systematic screen for Escherichia coli mutants that constitutively express the SOS regulon. We identified mutant strains with transposon insertions in 42 genes that caused increased expression from a dinD1::lacZ reporter construct. Most of these also displayed significant increases in basal levels of RecA protein, confirming an effect on the SOS system. As expected, this collection includes genes, such as lexA, dam, rep, xerCD, recG, and polA, which have previously been shown to cause an SOS constitutive phenotype when inactivated. The collection also includes 28 genes or open reading frames that were not previously identified as SOS constitutive, including dcd, ftsE, ftsX, purF, tdcE, and tynA. Further study of these SOS constitutive mutants should be useful in understanding the multiple causes of endogenous DNA damage. This study also provides a quantitative comparison of the extent of SOS expression caused by inactivation of many different genes in a common genetic background.     

Genetic interactions of DNA repair pathways in the pathogen Neisseria meningitidis

The current increase in the incidence and severity of infectious diseases mandates improved understanding of the basic biology and DNA repair profiles of virulent microbes. In our studies of the major pathogen and model organism Neisseria meningitidis, we constructed a panel of mutants inactivating genes involved in base excision repair, mismatch repair, nucleotide excision repair (NER), translesion synthesis, and recombinational repair pathways. The highest spontaneous mutation frequency among the N. meningitidis single mutants was found in the MutY-deficient strain as opposed to mutS mutants in Escherichia coli, indicating a role for meningococcal MutY in antibiotic resistance development. Recombinational repair was recognized as a major pathway counteracting methyl methanesulfonate-induced alkylation damage in the N. meningitidis. In contrast to what has been shown in other species, meningococcal NER did not contribute significantly to repair of alkylation-induced DNA damage, and meningococcal recombinational repair may thus be one of the main pathways for removal of abasic (apurinic/apyrimidinic) sites and strand breaks in DNA. Conversely, NER was identified as the main meningococcal defense pathway against UV-induced DNA damage. N. meningitidis RecA single mutants exhibited only a moderate decrease in survival after UV exposure as opposed to E. coli recA strains, which are extremely UV sensitive, possibly reflecting the lack of a meningococcal SOS response. In conclusion, distinct differences between N. meningitidis and established DNA repair characteristics in E. coli and other species were identified.     

A homologue of the Rad18 postreplication repair gene is required for DNA damage responses throughout the fission yeast cell cycle

Cells activate DNA repair pathways and cell cycle checkpoints when they suffer damage to their genome. They also activate tolerance pathways that facilitate survival. In Escherichia coli, a mechanism known as postreplication repair (PRR) is used to bypass lesions that would otherwise present a physical block to DNA polymerase. PRR has also been proposed to occur in eukaryotic cells, although the partitioning of DNA synthesis to a discrete S-phase would suggest that it is only operative within a defined period of the cell cycle. Eukaryotic PRR has been most extensively studied in the budding yeast Saccharomyces cerevisiae. Two important genes for components of this repair pathway are RAD6, which encodes an ubiquitin-conjugating enzyme, and RAD18, which encodes a RING-finger protein and forms a heterodimer with Rad6p. Rad18p can also bind to DNA. We report here the identification of the Schizosaccharomyces pombe homologue of RAD18, which we have denoted rhp18. rhp18 mutants are hypersensitive to DNA-damaging agents, but show this hypersensitivity throughout the cell cycle. rhp18 mutants are characterised by a longer than usual DNA damage checkpoint arrest that is required for their residual viability following irradiation. Genetic analyses show that rhp18 controls a unique DNA damage repair/tolerance pathway that extends beyond the requirement to tolerate damage during S-phase, suggesting a broader definition of the function of this eukaryotic PRR protein.     

C. elegans FANCD2 responds to replication stress and functions in interstrand cross-link repair

One of the least well understood DNA repair processes in cells is the repair of DNA interstrand cross-links (ICLs) which present a major obstacle to DNA replication and must be repaired or bypassed to allow fork progression. Fanconi anemia (FA) is an inherited genome instability syndrome characterized by hypersensitivity to ICL damage. Central to the FA repair pathway is FANCD2 that is mono-ubiquitylated in response to replication stress and ICL damage through the action of the FA core complex and its E3-ubiquitin ligase subunit, FANCL. In its mono-ubiquitylated form FANCD2 is recruited to repair foci where it is believed to somehow coordinate ICL repair and restart of impeded replication forks. However, the precise mechanism through which the FA pathway and mono-ubiquitylation of FANCD2 promotes ICL repair remains unclear. Here we report on a functional homologue of FANCD2 in C. elegans (FCD-2). Although fcd-2 mutants are homozygous viable, they are exquisitely sensitive to ICL-inducing agents, but insensitive to ionizing radiation (IR). fcd-2 is dispensable for meiotic recombination and activation of the S-phase checkpoint, indicating that ICL sensitivity is likely due to a repair rather than a signalling defect. Indeed, we show that FCD-2 is mono-ubiquitylated in response to ICL damage and is recruited to nuclear repair foci. Consistent with the sensitivity of fcd-2 mutants, FCD-2 focus formation is induced in response to ICL damage and replication stress, but not following IR, suggesting that FCD-2 responds to lesions that block DNA replication and not DNA double strand breaks per se. The realization that the FA pathway is conserved in a genetically tractable model system will permit the comprehensive analysis of the interplay between the FA, homologous recombination (HR), translesion synthesis (TLS) and nucleotide excision repair (NER) pathways, critical to the understanding of ICL repair.     

Checkpoint adaptation precedes spontaneous and damage-induced genomic instability in yeast

Despite the fact that eukaryotic cells enlist checkpoints to block cell cycle progression when their DNA is damaged, cells still undergo frequent genetic rearrangements, both spontaneously and in response to genotoxic agents. We and others have previously characterized a phenomenon (adaptation) in which yeast cells that are arrested at a DNA damage checkpoint eventually override this arrest and reenter the cell cycle, despite the fact that they have not repaired the DNA damage that elicited the arrest. Here, we use mutants that are defective in checkpoint adaptation to show that adaptation is important for achieving the highest possible viability after exposure to DNA-damaging agents, but it also acts as an entrée into some forms of genomic instability. Specifically, the spontaneous and X-ray-induced frequencies of chromosome loss, translocations, and a repair process called break-induced replication occur at significantly reduced rates in adaptation-defective mutants. This indicates that these events occur after a cell has first arrested at the checkpoint and then adapted to that arrest. Because malignant progression frequently involves loss of genes that function in DNA repair, adaptation may promote tumorigenesis by allowing genomic instability to occur in the absence of repair.     

DNA repair mutants defining G2 checkpoint pathways in Schizosaccharomyces pombe.

We have tested mutants corresponding to 20 DNA repair genes of the fission yeast Schizosaccharomyces pombe for their ability to arrest in G2 after DNA damage. Of the mutants tested, four are profoundly defective in this damage dependent G2 arrest. In addition, these four mutants are highly sensitive to a transient inhibition of DNA synthesis by hydroxyurea. This suggests that the pathway responsible for the recognition of DNA damage and the subsequent mitotic arrest, shares many functions with the mechanism that controls the dependency of mitosis on the completion of S phase. The phenotype of these checkpoint rad mutants in wee mutant backgrounds indicate that the G2 arrest response is mediated either through, or in parallel with, the activity of the cdc2 gene product.     

Emerging models for DNA repair: Dictyostelium discoideum as a model for nonhomologous end-joining

DNA double strand breaks (DSBs) are a particularly cytotoxic variety of DNA lesion that can be repaired by homologous recombination (HR) or nonhomologous end-joining (NHEJ). HR utilises sequences homologous to the damage DNA template to facilitate repair. In contrast, NHEJ does not require homologous sequences for repair but instead functions by directly re-joining DNA ends. These pathways are critical to resolve DSBs generated intentionally during processes such as meiotic and site-specific recombination. However, they are also utilised to resolve potentially pathological DSBs generated by mutagens and errors during DNA replication. The importance of DSB repair is underscored by the findings that defects in these pathways results in chromosome instability that contributes to a variety of disease states including malignancy. The general principles of NHEJ are conserved in eukaryotes. As such, relatively simple model organisms have been instrumental in identifying components of these pathways and providing a mechanistic understanding of repair that has subsequently been applied to vertebrates. However, certain components of the NHEJ pathway are absent or show limited conservation in the most commonly used invertebrate models exploited to study DNA repair. Recently, however, it has become apparent that vertebrate DNA repair pathway components, including those involved in NHEJ, are unusually conserved in the amoeba Dictyostelium discoideum. Traditionally, this genetically tractable organism has been exploited to study the molecular basis of cell type specification, cell motility and chemotaxis. Here we discuss the use of this organism as an additional model to study DNA repair, with specific reference to NHEJ.