Evidence for upregulated repair of oxidatively induced DNA damage in human colorectal cancer

Carcinogenesis may involve overproduction of oxygen-derived species including free radicals, which are capable of damaging DNA and other biomolecules in vivo. Increased DNA damage contributes to genetic instability and promote the development of malignancy. We hypothesized that the repair of oxidatively induced DNA base damage may be modulated in colorectal malignant tumors, resulting in lower levels of DNA base lesions than in surrounding pathologically normal tissues. To test this hypothesis, we investigated oxidatively induced DNA damage in cancerous tissues and their surrounding normal tissues of patients with colorectal cancer. The levels of oxidatively induced DNA lesions such as 4,6-diamino-5-formamidopyrimidine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 8-hydroxyguanine and (5'S)-8,5'-cyclo-2'-deoxyadenosine were measured by gas chromatography/isotope-dilution mass spectrometry and liquid chromatography/isotope-dilution tandem mass spectrometry. We found that the levels of these DNA lesions were significantly lower in cancerous colorectal tissues than those in surrounding non-cancerous tissues. In addition, the level of DNA lesions varied between colon and rectum tissues, being lower in the former than in the latter. The results strongly suggest upregulation of DNA repair in malignant colorectal tumors that may contribute to the resistance to therapeutic agents affecting the disease outcome and patient survival. The type of DNA base lesions identified in this work suggests the upregulation of both base excision and nucleotide excision pathways. Development of DNA repair inhibitors targeting both repair pathways may be considered for selective killing of malignant tumors in colorectal cancer.     

Measurement of oxidatively induced DNA damage and its repair,by mass spectrometric techniques

Abstract

Oxidatively induced damage caused by free radicals and other DNA-damaging agents generate a plethora of products in the DNA of living organisms. There is mounting evidence for the involvement of this type of damage in the etiology of numerous diseases including carcinogenesis. For a thorough understanding of the mechanisms, cellular repair, and biological consequences of DNA damage, accurate measurement of resulting products must be achieved. There are various analytical techniques, with their own advantages and drawbacks, which can be used for this purpose. Mass spectrometric techniques with isotope dilution, which include gas chromatography (GC) and liquid chromatography (LC), provide structural elucidation of products and ascertain accurate quantification, which are absolutely necessary for reliable measurement. Both gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS), in single or tandem versions, have been used for the measurement of numerous DNA products such as sugar and base lesions, 8,5’-cyclopurine-2’-deoxynucleosides, base-base tandem lesions, and DNA-protein crosslinks, in vitro and in vivo. This article reviews these techniques and their applications in the measurement of oxidatively induced DNA damage and its repair.     

The controlling role of ATM in homologous recombinational repair of DNA damage

The human genetic disorder ataxia telangiectasia (A-T), caused by mutation in the ATM gene, is characterized by chromosomal instability, radiosensitivity and defective cell cycle checkpoint activation. DNA double-strand breaks (dsbs) persist in A-T cells after irradiation, but the underlying defect is unclear. To investigate ATM's interactions with dsb repair pathways, we disrupted ATM along with other genes involved in the principal, complementary dsb repair pathways of homologous recombination (HR) or non-homologous end-joining (NHEJ) in chicken DT40 cells. ATM(-/-) cells show altered kinetics of radiation-induced Rad51 and Rad54 focus formation. Ku70-deficient (NHEJ(-)) ATM(-/-) chicken DT40 cells show radiosensitivity and high radiation-induced chromosomal aberration frequencies, while Rad54-defective (HR(-)) ATM(-/-) cells show only slightly elevated aberration levels after irradiation, placing ATM and HR on the same pathway. These results reveal that ATM defects impair HR-mediated dsb repair and may link cell cycle checkpoints to HR activation.     

Differential requirements for RAD51 in Physcomitrella patens and Arabidopsis thaliana development and DNA damage repair

RAD51, the eukaryotic homolog of the bacterial RecA recombinase, plays a central role in homologous recombination (HR) in yeast and animals. Loss of RAD51 function causes lethality in vertebrates but not in other animals or in the flowering plant Arabidopsis thaliana, suggesting that RAD51 is vital for highly developed organisms but not for others. Here, we found that loss of RAD51 function in the moss Physcomitrella patens, a plant of less complexity, caused a significant vegetative phenotype, indicating an important function for RAD51 in this organism. Moreover, loss of RAD51 caused marked hypersensitivity to the double-strand break-inducing agent bleomycin in P. patens but not in Arabidopsis. Therefore, HR is used for somatic DNA damage repair in P. patens but not in Arabidopsis. These data imply fundamental differences in the use of recombination pathways between plants. Moreover, these data demonstrate that the importance of RAD51 for viability is independent of taxonomic position or complexity of an organism. The involvement of HR in DNA damage repair in the slowly evolving species P. patens but not in fast-evolving Arabidopsis suggests that the choice of the recombination pathway is related to the speed of evolution in plants.     

Nitric oxide restrain root growth by DNA damage induced cell cycle arrest in Arabidopsis thaliana

Nitric oxide (NO) participates in the regulation of diverse functions in plant cells. However, different NO concentrations may trigger different pathways during the plant development. At basal levels of NO, plants utilize the NO signaling transduction pathway to facilitate plant growth and development, whereas higher concentrations trigger programmed cell death (PCD). Our results show that NO lower than the levels causing PCD, but higher than the basal levels induce DNA damage in root cells in Arabidopsis as witnessed by a reduction in root growth, rather than cell death, since cells retain the capacity to differentiate root hairs. The decrease in meristematic cells and increase in DNA damage signals in roots in responses to NO are in a dose dependent manner. The restraint of root growth is due to cell cycle arrest at G1 phase which is caused by NO induced DNA damage, besides a second arrest at G2/M existed in NO supersensitive mutant cue1. The results indicate that NO restrain root growth via DNA damage induced cell cycle arrest.     

RAD51 paralogs: roles in DNA damage signalling, recombinational repair and tumorigenesis

Chromosomal double-strand breaks (DSBs) have the potential to permanently arrest cell cycle progression and endanger cell survival. They must therefore be efficiently repaired to preserve genome integrity and functionality. Homologous recombination (HR) provides an important error-free mechanism for DSB repair in mammalian cells. In addition to RAD51, the central recombinase activity in mammalian cells, a family of proteins known as the RAD51 paralogs and consisting of five proteins (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3), play an essential role in the DNA repair reactions through HR. The RAD51 paralogs act to transduce the DNA damage signal to effector kinases and to promote break repair. However, their precise cellular functions are not fully elucidated. Here we discuss recent advances in our understanding of how these factors mediate checkpoint responses and act in the HR repair process. In addition, we highlight potential functional similarities with the BRCA2 tumour suppressor, through the recently reported links between RAD51 paralog deficiencies and tumorigenesis triggered by genome instability.     

Genome-wide assessment of oxidatively generated DNA damage

Abstract

In the tide of science nouveau after the completion of genome projects of various species, there appeared a movement to understand an organism as a system rather than the sum of cells directed for certain functions. With the advent and spread of microarray techniques, systematic and comprehensive genome-wide approaches have become reasonably possible and more required on the investigation of DNA damage and the subsequent repair. The immunoprecipitation-based technique combined with high-density microarrays or next-generation sequencing is one of the promising methods to provide access to such novel research strategies. Oxygen is necessary for most of the life on earth for electron transport. However, reactive oxygen species are inevitably generated, giving rise to steady-state levels of DNA damage in the genome, that may cause mutations leading to cancer, ageing and degenerative diseases. Previously, we showed that there are many factors involved in the genomic distribution of oxidatively generated DNA damage including chromosome territory, and proposed this sort of research area as oxygenomics. Recently, RNA is also recognized as a target of this kind of modification.     

The DNA dioxygenase ALKBH2 protects Arabidopsis thaliana against methylation damage

The Escherichia coli AlkB protein (EcAlkB) is a DNA repair enzyme which reverses methylation damage such as 1-methyladenine (1-meA) and 3-methylcytosine (3-meC). The mammalian AlkB homologues ALKBH2 and ALKBH3 display EcAlkB-like repair activity in vitro, but their substrate specificities are different, and ALKBH2 is the main DNA repair enzyme for 1-meA in vivo. The genome of the model plant Arabidopsis thaliana encodes several AlkB homologues, including the yet uncharacterized protein AT2G22260, which displays sequence similarity to both ALKBH2 and ALKBH3. We have here characterized protein AT2G22260, by us denoted ALKBH2, as both our functional studies and bioinformatics analysis suggest it to be an orthologue of mammalian ALKBH2. The Arabidopsis ALKBH2 protein displayed in vitro repair activities towards methyl and etheno adducts in DNA, and was able to complement corresponding repair deficiencies of the E. coli alkB mutant. Interestingly, alkbh2 knock-out plants were sensitive to the methylating agent methylmethanesulphonate (MMS), and seedlings from these plants developed abnormally when grown in the presence of MMS. The present study establishes ALKBH2 as an important enzyme for protecting Arabidopsis against methylation damage in DNA, and suggests its homologues in other plants to have a similar function.     

DNA repair efficiency for ultraviolet induced damage

Ultraviolet radiation causes lesions in bacterial DNA which are repaired by several enzyme systems. Wide variations in the efficiency of repair for differentE. Coli strains are inadequately explained by a simple presence or absence of one or more repair systems. It is proposed that a major factor in the variations is the sensitivity of the repair systems themselves to ultraviolet induced interactions between proteins and the repair enzyme cistrons. An analytic approach is applied to pre-existing data to establish the numbers of thymine and cytosine bases in the repair cistrons, lending support to the model. The findings imply that bacteria will become sensitive to UV upon inhibition of one of four amino acids.     

Genome-wide assessment of oxidatively generated DNA damage

In the tide of science nouveau after the completion of genome projects of various species, there appeared a movement to understand an organism as a system rather than the sum of cells directed for certain functions. With the advent and spread of microarray techniques, systematic and comprehensive genome-wide approaches have become reasonably possible and more required on the investigation of DNA damage and the subsequent repair. The immunoprecipitation-based technique combined with high-density microarrays or next-generation sequencing is one of the promising methods to provide access to such novel research strategies. Oxygen is necessary for most of the life on earth for electron transport. However, reactive oxygen species are inevitably generated, giving rise to steady-state levels of DNA damage in the genome, that may cause mutations leading to cancer, ageing and degenerative diseases. Previously, we showed that there are many factors involved in the genomic distribution of oxidatively generated DNA damage including chromosome territory, and proposed this sort of research area as oxygenomics. Recently, RNA is also recognized as a target of this kind of modification.     

A comparison of two DNA base excision repair glycosylases from Arabidopsis thaliana

Plants contain the genes for both formamidopyrimidine-DNA glycosylase (FPG) and oxoguanine glycosylase (OGG). These enzymes play analogous roles in the base excision repair pathways of bacteria (FPG) and archaea, yeast, and mammals (OGG). Why have plants retained both genes? We tested one hypothesis by comparing the specificities of Arabidopsis FPG and OGG purified from Escherichia coli expression clones. Using depurinated DNA as substrate, the specific activity of Arabidopsis FPG was higher than that of Arabidopsis OGG. Using DNA oxidized by treatment with light in the presence of methylene blue, the specific activities of Arabidopsis FPG and OGG were equal. Using an oligonucleotide containing one oxoguanine (paired with C) and labeled with fluorescein, the specific activity of Arabidopsis OGG was greater than that of either FPG. The results support the hypothesis that genes for the two enzymes have been retained during evolution of plants for their specialized enzyme activities.     

DNA damage and repair in Arabidopsis thaliana as measured by the comet assay after treatment with different classes of genotoxins

We have studied the occurrence of the apoptosis phenomenon in the action of adriamycin (ADR) on human melanoma cells sensitive (ME18) and resistant (ME18/R) to ADR.

The study has shown that the intensity of apoptotic morphological changes noted in melanoma cells depended on the duration of the ADR treatment.

We have not observed any positive correlation between the induction of apoptosis and sensitivity to ADR.

We have used a fluorescence microscope and flow cytometer to evaluate apoptotic events in cells treated with ADR.     

The DNA of Arabidopsis thaliana

Molecular Genetics and Genomics - Arabidopsis thaliana is a small flowering plant of the mustard family. It has a four to five week generation time, can be self- or cross-pollinated and bears as...     

TOPBP1 takes RADical command in recombinational DNA repair

TOPBP1 is a key player in DNA replication and DNA damage signaling. In this issue, Moudry et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201507042) uncover a crucial role for TOPBP1 in DNA repair by revealing its requirement for RAD51 loading during repair of double strand breaks by homologous recombination.Proper replication and maintenance of the eukaryotic genome requires the involvement of the scaffolding protein TOPBP1. Over the last 20 years, studies in yeast, frog, and mammals have revealed conserved roles for TOPBP1 in initiation of DNA replication and activation of DNA damage signaling. TOPBP1 has been shown to assemble ternary protein complexes necessary to jump-start DNA replication or to initiate DNA damage signaling events by recognizing distinct phosphoproteins via its multiple BRCA1 C terminus (BRCT) domains (Fig. 1 D; Wardlaw et al., 2014). In this issue, Moudry et al. add to the list of crucial TOPBP1 roles in genome biology and reveal that TOPBP1 is also required for proper repair of double strand breaks (DSBs) by homologous recombination (HR).Open in a separate windowFigure 1.Involvement of TOPBP1 in HR-mediated repair. (A) The work by Moudry et al. (2016) supports a model in which TOPBP1 directs the action of PLK1 kinase toward RAD51, mediating a phosphorylation event that licenses a second phosphorylation event mediated by the casein kinase 2 (CK2) kinase. These phosphorylation events are believed to facilitate the loading of RAD51, which replaces RPA, at damage sites and favor HR. (B) TOPBP1 as a hub for coordinating the action of multiple kinases toward genome maintenance. (C) Speculative model for the mutually exclusive engagement of TOPBP1 in distinct cellular processes as a strategy for coordinating genome replication and DNA damage responses (see text for details). (D) Current understanding of how TOPBP1 and its yeast orthologue Dpb11 mediate the formation of ternary complexes for replication initiation, DNA damage signaling, and recombinational repair. In mammals, the indicated proteins have been shown to interact with TOPBP1, but the roles of most of those interactions remain unclear. For simplicity, some TOPBP1 interactions and their cofactors are not depicted.Moudry et al. (2016) report that depletion of TOPBP1 makes cells highly sensitive to the poly (ADP-ribose) polymerase inhibitor olaparib, a drug known to sensitize cells with an already dysfunctional HR machinery. In particular, olaparib hypersensitizes cells that carry mutations in the bona fide HR factors and tumor suppressors BRCA1 or BRCA2. In this work, the authors first identified TOPBP1 as a hit in a high-content RNAi screen for proteins whose depletion resulted in higher toxicity after olaparib treatment in osteosarcoma cells, which suggests that loss or inactivation of TOPBP1 predicts the response of cancer cells to this drug. Moudry et al. (2016) observed that RNAi-mediated knockdown of TOPBP1 in cancer cells treated with olaparib increased the level of DNA damage and induced DNA DSB markers. The researchers subsequently examined whether olaparib sensitivity reflected defective HR in TOPBP1-depleted cells by measuring HR activity through several parameters and confirmed that TOPBP1-depleted cells showed reduced HR activity.The HR process encompasses several phases, including end resection and chromatin loading of RPA and RAD51, which can be visualized by formation of microscopically detectable foci. Moudry et al. (2016) searched for which step of HR was compromised in cells depleted for TOPBP1 and found that DNA end resection, i.e., the processing of the 5′ recessed end that exposes a 3′ overhang used for homology search, seemed not to be affected, as evaluated by the amounts of single stranded DNA detected by BrdU incorporation under nondenaturing conditions. Interestingly, they found that the next key stage in HR, in which the RAD51 recombinase protein is loaded at these 3′ overhangs (Fig. 1 A), was greatly impaired, based on the assessment of the formation of RAD51 foci by microscopy and of the biochemical analysis of RAD51 accumulation on chromatin. Although the mechanism by which TOPBP1 promotes the loading of RAD51 remains unclear, the authors propose an interesting model in which TOPBP1 plays a scaffolding role to direct Polo-like Kinase 1 (PLK1), which phosphorylates RAD51 and facilitates its loading to DNA damage sites (Fig. 1 A; Yata et al., 2012). Consistent with this model, they show that TOPBP1 physically interacts with PLK1 and that depletion of TOPBP1 impairs PLK1-dependent RAD51 phosphorylation. Although more work is needed to prove that the TOPBP1–PLK1 interaction is required for this phosphorylation event, the results are exciting as they suggest another important functional link between TOPBP1 and a kinase. During DNA damage signaling, TOPBP1 plays an established role in activating the ATR kinase (Kumagai et al., 2006) and is believed to direct ATR’s action toward specific substrates. This latter function is best understood in yeast, in which TOPBP1/Dpb11 forms a ternary complex to direct ATR/Mec1 action to phosphorylate the downstream kinase Rad53. Interestingly, recent data from fission yeast also suggest that TOPBP1 interacts with yet another kinase, CDK, and directs its kinase action (Qu et al., 2013). The emerging scenario is that TOPBP1 may function as a scaffolding hub for controlling the action of distinct kinases to ensure genome integrity (Fig. 1 B).Although the work of Moudry et al. (2016) is the first to show a clear role for TOPBP1 in RAD51 loading, studies in budding yeast have proposed links between the TOPBP1 orthologue Dpb11 and HR-mediated repair. It was shown that the temperature-sensitive dpb11-1 mutant displays a sensitivity to DNA damage that is not further increased by deletion of RAD51, suggesting that Dpb11 functions in HR repair (Ogiwara et al., 2006). In addition, other groups showed that TOPBP1/Dpb11 is required for DSB-induced mating-type switching and also reached the conclusion that TOPBP1/Dpb11 is required for HR-mediated repair of a DSB (Germann et al., 2011; Hicks et al., 2011). These studies provided compelling evidence that the role for TOPBP1/Dpb11 in DSB repair is independent of its roles in replication initiation and DNA damage signaling. In humans, there also is evidence pointing to potential roles for TOPBP1 in DNA repair, as depletion of TOPBP1 was found to increase sensitivity to ionizing radiation and lead to defective DSB repair by HR (Morishima et al., 2007).The new set of results provided by Moudry et al. (2016) clearly place TOPBP1 at the center stage of HR-mediated repair in what seems to be yet another key and evolutionarily conserved role for TOPBP1, in addition to replication initiation and DNA damage signaling. An intriguing and unanswered question relates to defining the evolutionary benefit conferred by maintaining these crucial roles in the same protein. It is tempting to speculate that having a single protein module in command of key licensing events helps ensure the ordered and mutually exclusive execution of distinct cellular processes (Fig. 1 C). This is a particularly attractive and well-suited idea for the established role of DNA damage signaling in inhibiting origin firing during DNA replication. Sequestration of TOPBP1 into a complex involved in DNA damage signaling would help ensure that replication initiation is inhibited. Consistent with this hypothesis, it is established in yeast that the same BRCT domains involved in replication initiation are also required for DNA damage signaling. In addition, it was recently shown that competition between DNA damage signaling proteins and DNA repair factors for binding to the BRCT domains of TOPBP1/Dpb11 is a mechanism to remove TOPBP1/Dpb11 from a pro-DNA damage signaling complex, resulting in dampening of DNA damage signaling (Ohouo et al., 2013; Cussiol et al., 2015). It will be exciting to further explore this competition-based regulatory mechanism in human cells, as well as in the coordination of DNA damage signaling with DNA repair. In this direction, it is crucial that the precise molecular mechanism by which TOPBP1 promotes HR repair is elucidated, including defining which TOPBP1 BRCT domains are required and which factors they are binding to favor RAD51 loading or other pro-HR functions. Through truncation mutation analyses, Moudry et al. (2016) show that the specific BRCT domains 7/8 of TOPBP1 are essential for TOPBP1’s role in promoting HR. However, it remains unclear how this is accomplished mechanistically.To make the scenario even more complicated, TOPBP1 is known to physically interact with an extensive network of repair factors, including, but not limited to, BRCA1, 53BP1, MRN, FANCJ, and BLM (Greenberg et al., 2006; Wardlaw et al., 2014). This points to an extremely complex system by which TOPBP1 could be coordinating the action of a range of repair factors and repair pathways (Fig. 1 D). It would not be surprising if TOPBP1 was found to be key for the regulation of other steps in HR-mediated repair as well as other repair pathways in response to varied types of genotoxic insults, including DNA replication stress. In yeast, the interaction between TOPBP1/Dpb11 and the repair scaffold Slx4 provides an additional example of the rich range of possibilities by which TOPBP1/Dpb11 functions in DNA repair. In addition to sequestering TOPBP1/Dpb11 and dampening DNA damage signaling (Ohouo et al., 2013; Cussiol et al., 2015), the Slx4–TOPBP1/Dpb11 interaction was recently found to control DNA end resection (Dibitetto et al., 2015) and was proposed to affect the late step of resolution of repair intermediates (Gritenaite et al., 2014). The TOPBP1–SLX4 interaction is conserved in humans; however, it remains unclear how this interaction impacts DNA repair in higher eukaryotes. Moreover, whereas in yeast it is possible to clearly define a pro-DNA damage signaling complex and a pro-recombinational repair complex (Fig. 1 D), in mammals the scenario is more complex and it is currently unclear what the precise contributions of different TOPBP1 interactions are in DNA damage signaling and/or recombinational repair. Finally, because the ATR kinase is expected to regulate several DNA repair factors, it is likely that the ATR-activating function of TOPBP1 plays important roles in some aspect of DNA repair. A major experimental avenue to explore this possibility and improve our understanding of the other roles for TOPBP1 in DNA repair will be the generation of separation-of-function mutants that do not interfere with DNA replication or DNA damage signaling.Following the findings reported by Moudry et al. (2016), it is interesting to speculate on the implications of understanding TOPBP1’s role in HR-mediated repair for cancer research and treatment. Little is known about the role of TOPBP1 in carcinogenesis. It was found that TOPBP1 expression and subcellular localization are altered in a subset of breast cancer samples (Going et al., 2007; Liu et al., 2009; Forma et al., 2012) and Moudry et al. (2016) also report altered TOPBP1 protein expression in ovarian cancers, although at modest frequencies. Nonetheless, as we learn more about TOPBP1 mechanisms of action in HR, it is possible that it may become an important target for manipulating the HR response by using small molecules such as Calcein AM, which targets BRCT domains 7/8 of TOPBP1 (Chowdhury et al., 2014) and was shown by Moudry et al. (2016) to impair HR. Concerning the finding that TOPBP1 plays a pro-HR function very much like BRCA1 and BRCA2, whose genes are most frequently mutated in ovarian and breast cancers, it is intriguing that although TOPBP1 mutations have been found in cancers (Rebbeck et al., 2009; Forma et al., 2013), they are relatively infrequent and are likely not driver mutations. If TOPBP1 plays a key role in RAD51 loading, which is the step severely perturbed in BRCA1- or BRCA2-mutated cancer cells, it is not clear how more cancer-driving mutations have not been identified in TOPBP1. One possibility is that TOPBP1 mutations affecting TOPBP1’s pro-HR function also affect DNA replication and DNA damage signaling and impair the replicative capacity of cancer cells. Disentangling potential antagonistic roles for TOPBP1 in both suppressing and supporting tumorigenesis could lead to exciting new directions to study this complex multifunctional protein and to potentially develop new therapeutic strategies.     

Measurement of oxidatively generated base damage in cellular DNA

This survey focuses on the critical evaluation of the main methods that are currently available for monitoring single and complex oxidatively generated damage to cellular DNA. Among chromatographic methods, HPLC-ESI-MS/MS and to a lesser extent HPLC-ECD which is restricted to a few electroactive nucleobases and nucleosides are appropriate for measuring the formation of single and clustered DNA lesions. Such methods that require optimized protocols for DNA extraction and digestion are sensitive enough for measuring base lesions formed under conditions of severe oxidative stress including exposure to ionizing radiation, UVA light and high intensity UVC laser pulses. In contrast application of GC-MS and HPLC-MS methods that are subject to major drawbacks have been shown to lead to overestimated values of DNA damage. Enzymatic methods that are based on the use of DNA repair glycosylases in order to convert oxidized bases into strand breaks are suitable, even if they are far less specific than HPLC methods, to deal with low levels of single modifications. Several other methods including immunoassays and (32)P-postlabeling methods that are still used suffer from drawbacks and therefore are not recommended. Another difficult topic is the measurement of oxidatively generated clustered DNA lesions that is currently achieved using enzymatic approaches and that would necessitate further investigations.     

DNA damage induced nucleotide excision repair in Saccharomyces cerevisiae

Nucleotide excision repair (NER) is the most versatile and universal pathway of DNA repair that is capable of repairing virtually any damages other than a double strand break (DSB). This pathway has been shown to be inducible in several systems. However, question of a threshold and the nature of the damage that can signal induction of this pathway remain poorly understood. In this study it has been shown that prior exposure to very low doses of osmium tetroxide enhanced the survival of wild type Saccharomyces cerevisiae when the cells were challenged with UV light. Moreover, it was also found that osmium tetroxide treated rad3 mutants did not show enhanced survival indicating an involvement of nucleotide excision repair in the enhanced survival. To probe this further the actual removal of pyrimidine dimers by the treated and control cells was studied. Osmium tetroxide treated cells removed pyrimidine dimers more efficiently as compared to control cells. This was confirmed by measuring the in vitro repair synthesis in cell free extracts prepared from control and primed cells. It was found that the uptake of active ³²P was significantly higher in the plasmid substrates incubated with extracts of primed cells. This induction is dependent on de novo synthesis of proteins as cycloheximide treatment abrogated this response. The nature of induced repair was found to be essentially error free. Study conclusively shows that NER is an inducible pathway in Saccharomyces cerevisiae and its induction is dependent on exposure to a threshold of a genotoxic stress.     

Genetic characterization of late-flowering traits induced by DNA hypomethylation mutation in Arabidopsis thaliana

Arabidopsis DNA hypomethylation mutation, ddm1 , results in a variety of developmental abnormalities by slowly inducing heritable lesions at unlinked loci. Here, late-flowering traits observed at high frequencies in independently-established ddm1 lines were genetically characterized. In all of the four late-flowering lines examined the traits were dominant and mapped to the same chromosomal region, which is close or possibly identical to the FWA locus. The ddm1 -induced phenotypic onsets are apparently not random mutation events, but specific to a group of genes, suggesting the underlying epigenetic mechanism. The DNA methylation mutant provide useful system for identifying epigenetically-regulated genes important for plant development.     

DNA damage checkpoint and recombinational repair differentially affect the replication stress tolerance of smc6 mutants

DNA damage checkpoint and recombinational repair are both important for cell survival of replication stress. Because these two processes influence each other, isolation of their respective contributions is challenging. Research in budding yeast shows that removal of the DNA helicase Mph1 improves survival of cells with defective Smc5/6 complex under replication stress. mph1∆ is known to reduce the levels of recombination intermediates in smc6 mutants. Here, we show that mph1∆ also hyperactivates the Mec1 checkpoint. We dissect the effects of recombination regulation and checkpoint hyperactivation by altering the checkpoint circuitry to enhance checkpoint signaling without reducing recombination intermediate levels. We show that these approaches, similar to mph1∆, lead to better survival of smc6 cells upon transient replication stress, likely by ameliorating replication and chromosomal segregation defects. Unlike mph1∆, however, they do not suppress smc6 sensitivity to chronic stress. Conversely, reducing the checkpoint response does not impair survival of smc6 mph1∆ mutants under chronic stress. These results suggest a two-phase model in which smc6 mutant survival upon transient replication stress can be improved by enhancing Mec1 checkpoint signaling, whereas smc6 sensitivity to chronic stress can be overcome by reducing recombination intermediates.