Conditions Of DNA Damage Research Articles

A nucleolar role for the Fanconi anemia pathway protein, FANCI, in rDNA transcription

Fanconi anemia (FA) is a disease of DNA repair because proteins mutated in patients with FA are impaired in removing interstrand DNA crosslinks. We propose a new nucleolar role for the FA protein, FANCI, in ribosome biogenesis based on extensive preliminary data. Ribosome biogenesis is the process of making ribosomes that initiates in the cell nucleolus with the transcription and processing of the pre‐ribosomal RNA (rRNA) followed by the assembly of mature ribosomes. Immunoaffinity purification of FANCI from HeLa cell nuclear extract followed by mass spectrometry revealed co‐purification with nucleolar proteins required for ribosome biogenesis, including PES1. Using subcellular fractionation, we have found that FANCI is indeed enriched in the nucleolus. In particular, the deubiquitinated form of FANCI is by far the most predominant form of nucleolar FANCI, even under conditions of DNA damage by mitomycin C, which stimulate monoubiquitination of FANCI. Immunoprecipitation revealed that deubiquitinated FANCI, but not its binding partner in DNA repair, FANCD2, is co‐immunoprecipitated by the nucleolar protein, PES1. To probe a possible nucleolar function for FANCI, we depleted FANCI in human cells and observed reduced transcription by RNA polymerase I (RNAPI). In addition, FANCI is co‐immunoprecipitated by the large subunit of RNAPI, indicating a physical and functional role for FANCI in efficient pre‐rRNA transcription. Finally, our results demonstrate novel regulatory mechanisms by deubiquitinases on the ubiquitination state of FANCI in the nucleolus. While our model does not contest the well‐established underlying DNA repair defect observed in cells from FA patients, it does suggest a dual pathophysiology for FA that includes defects both in DNA repair and in ribosome biogenesis.Support or Funding InformationThis work is funded by the NIGMS and NCI.

Extracellular ATP protects endothelial cells against DNA damage.

Cell damage can lead to rapid release of ATP to extracellular space resulting in dramatic change in local ATP concentration. Evolutionary, this has been considered as a danger signal leading to adaptive responses in adjacent cells. Our aim was to demonstrate that elevated extracellular ATP or inhibition of ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1/CD39) activity could be used to increase tolerance against DNA-damaging conditions. Human endothelial cells, with increased extracellular ATP concentration in cell proximity, were more resistant to irradiation or chemically induced DNA damage evaluated with the DNA damage markers γH2AX and phosphorylated p53. In our rat models of DNA damage, inhibiting CD39-driven ATP hydrolysis with POM-1 protected the heart and lung tissues against chemically induced DNA damage. Interestingly, the phenomenon could not be replicated in cancer cells. Our results show that transient increase in extracellular ATP can promote resistance to DNA damage.

The Protein Arginine Methylase 5 (PRMT5/SKB1) Gene Is Required for the Maintenance of Root Stem Cells in Response to DNA Damage

Plant root stem cells and their surrounding microenvironment, namely the stem cell niche, are hypersensitive to DNA damage. However, the molecular mechanisms that help maintain the genome stability of root stem cells remain elusive. Here we show that the root stem cells in the skb1 (Shk1 kinase binding protein 1) mutant undergoes DNA damage-induced cell death, which is enhanced when combined with a lesion of the Ataxia-telangiectasia mutated (ATM) or the ATM/RAD3-related (ATR) genes, suggesting that the SKB1 plays a synergistically effect with ATM and ATR in DNA damage pathway. We also provide evidence that SKB1 is required for the maintenance of quiescent center (QC), a root stem cell niche, under DNA damage treatments. Furthermore, we report decreased and ectopic expression of SHORTROOT (SHR) in response to DNA damage in the skb1 root tips, while the expression of SCARECROW (SCR) remains unaffected. Our results uncover a new mechanism of plant root stem cell maintenance under DNA damage conditions that requires SKB1.

NAT10 regulates p53 activation through acetylating p53 at K120 and ubiquitinating Mdm2.

As a genome guardian, p53 maintains genome stability by arresting cells for damage repair or inducing cell apoptosis to eliminate the damaged cells in stress response. Several nucleolar proteins stabilize p53 by interfering Mdm2–p53 interaction upon cellular stress, while other mechanisms by which nucleolar proteins activate p53 remain to be determined. Here, we identify NAT10 as a novel regulator for p53 activation. NAT10 acetylates p53 at K120 and stabilizes p53 by counteracting Mdm2 action. In addition, NAT10 promotes Mdm2 degradation with its intrinsic E3 ligase activity. After DNA damage, NAT10 translocates to nucleoplasm and activates p53‐mediated cell cycle control and apoptosis. Finally, NAT10 inhibits cell proliferation and expression of NAT10 decreases in human colorectal carcinomas. Thus, our data demonstrate that NAT10 plays a critical role in p53 activation via acetylating p53 and counteracting Mdm2 action, providing a novel pathway by which nucleolar protein activates p53 as a cellular stress sensor.

Stem Cell Replicative Stress and Clonal Exhaustion

In humans, the process of aging is characterized by a gradual decline in the functional capacity of multiple tissues, either directly precipitating age-associated diseases or increasing the risk of an individual developing disease, such as in the case of persistent infections or cancer. In regenerating organs, the process of aging is likely driven by the progressive depletion of adult stem cell populations that are responsible for maintaining tissues throughout the lifetime of an organism. One mechanism thought to be a primary cause of progressive adult stem cell depletion with ongoing time, is the accumulation of DNA damage in the stem cell compartment and the subsequent response to this insult. As well as potentially driving the loss of adult stem cells, DNA damage in this cell population is the likely mechanism behind the sequential acquisition of transforming mutations that lead to malignant transformation. Critically, to date, no one has identified the universal physiologic source of DNA damage in adult stem cells that leads to age-associated functional decline and transformation. An in depth interrogation of the mechanism via which adult stem cells acquire DNA damage under physiologic conditions and subsequently respond to this insult is therefore warranted in order to understand the aging process and how this is a critical factor in determining risk of developing cancer and other age-associated diseases. Historically, almost all studies of DNA damage and it's relevance to aging of stem cells either compare young versus aged cells and/or characterize the DNA damage response to artificial experimental agonists such as ionizing radiation, chemotherapy, serial bone marrow transplantation or in vitro culture. Although these approaches have been valuable in furthering our understanding of the relationship between DNA damage and stem cell biology, their physiologic relevance is debatable. The comparison of young versus aged cells assesses the consequences of cellular aging as opposed to the cause of aging, and non-physiologic experimental conditions of DNA damage evaluate a cellular response to an agonist of a type and magnitude that adult stem cells are unlikely to encounter in a normal lifetime. We have recently developed an in vivo model of DNA damage in hematopoietic stem cells (HSCs), which is precipitated by exposure of mice to agonists that mimic physiologic stress such as infection and chronic blood loss. These stress agonists drive HSCs out of their homeostatic quiescent status, resulting in de novo DNA damage as a consequence of increased replicative stress associated with dynamic changes in HSC energy metabolism. Importantly, this stress-induced DNA damage results in a phenotype of cumulative HSC attrition and a myeloid differentiation bias, which is akin to accelerated aging. In the setting of a clinically relevant mouse model of defective DNA repair (Fanconi anemia), stress hematopoiesis leads to a premature collapse of the entire hematopoietic system, fully recapitulating the progression of this disease in Fanconi anemia patients. Such a model is an ideal platform to study the response of HSCs to physiologic DNA damage and will allow us to better understand how environmental stress stimuli such as infections can impact upon both the rate of aging of tissues and the incidence of malignant transformation. This may have important clinical implications relevant to the study of age-related hematopoietic defects in patients. Disclosures No relevant conflicts of interest to declare.

Reciprocal regulation of p53 and NF-κB by diacylglycerol kinase ζ

Diacylglycerol kinase (DGK) participates in lipid mediated-signal transduction. It phosphorylates diacylglycerol (DG) to phosphatidic acid (PA), thereby regulating the balanced control of these second messenger actions. Previous reports have described that one DGK family, DGKζ, is closely involved in stress responses under various conditions. Cellular stress response, a physiological process enabling cells to cope with an altered environment, is finely tuned through various signaling cascades and their molecular crosstalk. The major components of stress response are p53 and NF-κB. p53 generally serves as a proapoptotic transcriptional factor, whereas NF-κB promotes resistance to programmed cell death under most circumstances. Recent studies have suggested that DGKζ facilitates p53 degradation in cytoplasm through ubiquitin proteasome system and that DGKζ deletion upregulates p53 protein levels under basal and DNA-damage conditions. Counter-intuitively, however, DGKζ deletion suppresses p53 transcriptional activity despite increased p53 levels. In contrast, DGKζ knockdown engenders enhancement of NF-κB pathway in response to cytokines such as TNF-α and IL-1β. In response to these cytokines, DGKζ downregulation accelerates phosphorylation of the p65 subunit and its nuclear translocation, thereby enhancing NF-κB transcriptional activity. Furthermore, DGKζ deficiency is shown to promote increased association of p65 subunit with the transcriptional cofactor CBP. It is particularly interesting that this association is observed even under basal conditions in the absence of stimulation. These findings suggest that DGKζ plays a role in sequestration of the limiting pool of CBP/p300 between the NF-κB p65 subunit and p53, and that DGKζ downregulation shifts CBP/p300 toward the NF-κB subunit to regulate reciprocally antagonistic phenotypes of these transcription factors.

Purification and Characterization of Recombinant Deinococcus radiodurans RNA Polymerase.

The radioresistant bacterium Deinococcus radiodurans is one of the most interesting models for studies of cell stress resistance. Analysis of the mechanisms of gene expression in D. radiodurans revealed some specific features of the transcription apparatus that might play a role in cell resistance to DNA-damaging conditions. In particular, RNA polymerase from D. radiodurans forms unstable promoter complexes and during transcription elongation has a much higher rate of RNA cleavage than RNA polymerase from Escherichia coli. Analysis of the structure and functions of D. radiodurans RNA polymerase is complicated due to the absence of convenient genetic systems for making mutations in the RNA polymerase genes and difficulties with enzyme purification. In this work, we developed a system for expression of D. radiodurans RNA polymerase in E. coli cells. We obtained an expression vector encoding all core RNA polymerase subunits and defined optimal conditions for the expression and purification of the RNA polymerase. It was found that D. radiodurans RNA polymerase has much higher rates of RNA cleavage than E. coli RNA polymerase under a wide range of conditions, including variations in the concentration of catalytic magnesium ions and pH values of the reaction buffer. The expression system can be used for further studies of the RNA cleavage reaction and the mechanisms of transcription regulation in D. radiodurans, including analysis of mutant RNA polymerase variants.

Stress-mediated Sin3B activation leads to negative regulation of subset of p53 target genes.

The multiprotein SWI-independent 3 (Sin3)-HDAC (histone deacetylase) corepressor complex mediates gene repression through its interaction with DNA-binding factors and recruitment of chromatin-modifying proteins on to the promoters of target gene. Previously, an increased expression of Sin3B and tumour suppressor protein, p53 has been established upon adriamycin treatment. We, now provide evidence that Sin3B expression is significantly up-regulated under variety of stress conditions and this response is not stress-type specific. We observed that Sin3B expression is significantly up-regulated both at transcript and at protein level upon DNA damage induced by bleomycin drug, a radiomimetic agent. This increase in Sin3B expression upon stress is found to be p53-dependent and is associated with enhanced interaction of Sin3B with Ser(15) phosphorylated p53. Binding of Sin3-HDAC repressor complex on to the promoters of p53 target genes influences gene regulation by altering histone modifications (H3K9me3 and H3K27me3) at target genes. Furthermore, knockdown of Sin3B by shRNA severely compromises p53-mediated gene repression under stress conditions. Taken together, these results suggest that stress-induced Sin3B activation is p53-dependent and is essential for p53-mediated repression of its selective target genes. The present study has an implication in understanding the transrepression mechanism of p53 under DNA damaging conditions.

SUB1 Plays a Negative Role during Starvation Induced Sporulation Program in Saccharomyces cerevisiae.

Saccharomyces cerevisiae Sub1 is involved in several cellular processes such as, transcription initiation, elongation, mRNA processing and DNA repair. It has also been reported to provide cellular resistance during conditions of oxidative DNA damage and osmotic stress. Here, we report a novel role of SUB1 during starvation stress-induced sporulation, which leads to meiosis and spore formation in diploid yeast cells. Deletion of SUB1 gene significantly increased sporulation efficiency as compared to the wild-type cells in S288c genetic background. Whereas, the sporulation functions of the sub1(Y66A) missense mutant were similar to Sub1. SUB1 transcript and protein levels are downregulated during sporulation, in highly synchronized and sporulation proficient wild-type SK1 cells. The changes in Sub1 levels during sporulation cascade correlate with the induction of middle sporulation gene expression. Deletion of SUB1 increased middle sporulation gene transcript levels with no effect on their induction kinetics. In wild-type cells, Sub1 associates with chromatin at these loci in a temporal pattern that correlates with their enhanced gene expression seen in sub1Δ cells. We show that SUB1 genetically interacts with HOS2, which led us to speculate that Sub1 might function with Set3 repressor complex during sporulation. Positive Cofactor 4, human homolog of Sub1, complemented the sub1Δ sporulation phenotype, suggesting conservation of function. Taken together, our results suggest that SUB1 acts as a negative regulator of sporulation.

Role of mTOR-Chk1 in enhancing DNA-damaging therapy

Mechanistic target of rapamycin (mTOR) is an atypical Ser/Thr protein kinase that acts as a cellular hub integrating cell growth signals and nutrient availability to promote cell survival.1 mTOR is the catalytic component of 2 functionally distinct complexes (mTORC1 and mTORC2), and belongs to the family of phosphatidyl-inositol(3)-like kinases (PIKKs) together with ATM, ATR and DNA-PKcs. The latter 3 kinases have well established roles in the DNA damage response (DDR) – the signaling network activated by DNA damage to promote cell survival by enhancing DNA repair and delaying cell cycle progression in order to maximize repair. However, the participation of mTOR in these pathways has only recently been recognized, aided by the generation and availability of ATP-competitive mTOR kinase inhibitors. A better understanding of the regulation of the DDR is not only vital to understand aspects of tumor development, but is also important to improve DNA damage-based cancer therapies by targeting specific aspects, such as mTOR signaling. mTOR may act to promote cell survival either by regulating DNA repair and/or cell cycle arrest. Indeed, recent work has demonstrated a role for mTOR kinase in DNA repair through the regulation of FANCD2, a key protein involved in the repair of double-strand DNA breaks.2,3 Our recent work in Oncotarget4 also implicates mTOR in control of the cell cycle arrest aspect of the DDR. We found that mTOR is required for efficient establishment of S and G2/M cell cycle arrest after DNA damage, since ablation of mTOR resulted in a reduction in the percentage of cells in S and G2/M. Interestingly, we found that mTORC2 was required for the DNA damage-induced activation of Chk1, a key regulator of cell cycle arrest. We found that mTOR inhibition significantly inhibited all 3 DNA damage-induced phosphorylations of Chk1 (S296, S317 and S345) and reduced the production of Chk1 protein. Since mTOR inhibition consistently ablated DNA damage-induced Chk1 phosphorylation under all conditions tested, whereas inhibition of protein levels was cell type specific, this suggests that there may be distinct mechanisms by which mTOR controls Chk1 and that phosphorylation of Chk1 may be of primacy. Exactly how mTOR might regulate Chk1 activity is an open question. One likely candidate could be via ATR, since its ability to phosphorylate Chk1 at Ser317 and Ser345 leading to autophosphorylation at Ser296 is well established. Alternatively, the effects on Chk1 might involve one of the substrates of mTORC2. Our results support accumulating evidence from studies in yeast implicating TORC2 in survival under conditions of DNA damage and in genome stability.5 Since approval of the rapalog, Everolimus, in combination with hormone therapies for breast cancer, the potential of mTOR inhibitors to overcome resistance to current therapies has gained increasing interest. Indeed numerous publications have demonstrated that mTOR inhibition can also enhance radio- or chemotherapy-induced cell death in various cancer types. We also investigated the translational significance of our results and found that an mTOR kinase inhibitor enhanced DNA damage-induced cell death in HEK293 cells and a panel of breast cancer cell lines.4 While this suggests that mTOR kinase inhibitors may be useful therapeutically to augment DNA damage based therapies, it is likely that these effects will be tumor-type specific since similar augmentation was not found in renal and colon cancers.6,7 We have previously shown that an mTOR kinase inhibitor prevented p53-dependent cell death in renal carcinoma cells.6 It is known that mTORC1 contributes to DNA damage-induced p53 activation via multiple mechanisms, therefore it is likely that mTOR ablation leads to p53 suppression and inhibition of p53-dependent apoptosis in these renal cells. However, HEK293 cells have non-functional p53 and the panel of breast cancer cell lines that we tested displayed a variety of either wild-type, lost or mutated p53, but all still exhibited chemosensitivity following mTOR kinase inhibition. This suggests that these latter cells may rely on mTORC2-Chk1 signaling for survival. In conclusion, it is clear that mTOR regulates the DDR response by multiple mechanisms. Whether a given tumor type is made chemosensitive or chemoresistant by mTOR kinase inhibitors will depend on which aspect of mTOR signaling is altered during tumorigenesis. In this vein, it will be important to widen our understanding of mTOR targets following DNA damage in order to gain better insight into whether specific targeting of mTORC2 will be a useful adjuvant to DNA-damaging therapies.

JMJD5 interacts with p53 and negatively regulates p53 function in control of cell cycle and proliferation

JMJD5 is a Jumonji C domain-containing demethylase/hydroxylase shown to be essential in embryological development, osteoclastic maturation, circadian rhythm regulation and cancer metabolism. However, its role and underlying mechanisms in oncogenesis remain unclear. Here, we demonstrate that JMJD5 forms complex with the tumor suppressor p53 by interacting with p53 DNA-binding domain (DBD), and negatively regulates its activity. Downregulation of JMJD5 resulted in increased expression of multiple p53 downstream genes, such as the cell cycle inhibitor CDKN1A and DNA repair effector P53R2, only in p53-proficient lung cancer cells. Upon DNA damage, the JMJD5–p53 association decreased, and thereby, promoted p53 recruitment to the target genes and stimulated its transcriptional activity. Furthermore, JMJD5 facilitated the cell cycle progression in a p53-dependent manner under both normal and DNA damage conditions. Depletion of JMJD5 inhibited cell proliferation and enhanced adriamycin-induced cell growth suppression in the presence of p53. Collectively, our results reveal that JMJD5 is a novel binding partner of p53 and it functions as a positive modulator of cell cycle and cell proliferation mainly through the repression of p53 pathway. Our study extends the mechanistic understanding of JMJD5 function in cancer development and implicates JMJD5 as a potential therapeutic target for cancer.

MDM4/HIPK2/p53 cytoplasmic assembly uncovers coordinated repression of molecules with anti-apoptotic activity during early DNA damage response.

The p53 inhibitor, MDM4 (MDMX) is a cytoplasmic protein with p53-activating function under DNA damage conditions. Particularly, MDM4 promotes phosphorylation of p53 at Ser46, a modification that precedes different p53 activities. We investigated the mechanism by which MDM4 promotes this p53 modification and its consequences in untransformed mammary epithelial cells and tissues. In response to severe DNA damage, MDM4 stimulates p53Ser46P by binding and stabilizing serine–threonine kinase HIPK2. Under these conditions, the p53-inhibitory complex, MDM4/MDM2, dissociates and this allows MDM4 to promote p53/HIPK2 functional interaction. Comparative proteomic analysis of DNA damage-treated cells versus -untreated cells evidenced a diffuse downregulation of proteins with anti-apoptotic activity, some of which were targets of p53Ser46P/HIPK2 repressive activity. Importantly, MDM4 depletion abolishes the downregulation of these proteins indicating the requirement of MDM4 to promote p53-mediated transcriptional repression. Consistently, MDM4-mediated HIPK2/p53 activation precedes HIPK2/p53 nuclear translocation and activity. Noteworthy, repression of these proteins was evident also in mammary glands of mice subjected to γ-irradiation and was significantly enhanced in transgenic mice overexpressing MDM4. This study evidences the flexibility of MDM2/MDM4 heterodimer, which allows the development of a positive activity of cytoplasmic MDM4 towards p53-mediated transcriptional function. Noteworthy, this activity uncovers coordinated repression of molecules with shared anti-apoptotic function which precedes active cell apoptosis and that are frequently overexpressed and/or markers of tumour phenotype in human cancer.

Investigating the Importance of DinB‐RecA Interactions to the Regulation of DinB's Activity and the Fidelity of the DNA Damage Response

The error prone DNA Polymerase IV, DinB, specializes in synthesizing DNA from a lesion‐containing DNA template, an activity that allows cell survival during DNA‐damaging conditions at a mutagenic cost. We have shown that there is direct physical interaction between DinB and Escherichia coli's main recombinase, RecA. We also have in vitro evidence suggesting that DinB•RecA interaction is conserved across many bacterial species, including human pathogens. Circular dichroism spectroscopy experiments show that the DinB•RecA complex undergoes a drastic conformational change compared to the single proteins alone. This is supported by the fact that DinB(C66A), a derivative with tight binding to RecA, and RecA copurify by size exclusion chromatography at a size that is much smaller than the size of the individual proteins, suggesting that compaction and tertiary structure changes are likely involved upon complex formation. Alteration of the physical interaction between DinB and RecA leads to changes in the mutagenic outcome in E. coli cells that are exposed to DNA damaging agents. Our current model is that the DinB•RecA complex not only alters the activity of DinB, but results in the sequestration of RecA from the lowest fidelity polymerase, DNA Polymerase V. The DinB•RecA complex, therefore, is central in controlling the fidelity of the DNA damage response.Source of Funding: R01 and its supplement from the National Institute of Health (NIH) Institute of General Medicine to V. Godoy

Yet another way that phage λ manipulates its Escherichia coli host: λrexB is involved in the lysogenic-lytic switch.

The life cycle of phage λ has been studied extensively. Of particular interest has been the process leading to the decision of the phage to switch from lysogenic to lytic cycle. The principal participant in this process is the λcI repressor, which is cleaved under conditions of DNA damage. Cleaved λcI no longer acts as a repressor, allowing phage λ to switch from its lysogenic to lytic cycle. The well-known mechanism responsible for λcI cleavage is the SOS response. We have recently reported that the Escherichia coli toxin-antitoxin mazEF pathway inhibits the SOS response; in fact, the SOS response is permitted only in E. coli strains deficient in the expression of the mazEF pathway. Moreover, in strains lysogenic for prophage λ, the SOS response is enabled by the presence of λrexB. λRexB had previously been found to inhibit the degradation of the antitoxin MazE, thereby preventing the toxic action of MazF. Thus, phage λ rexB gene not only safeguards the prophage state by preventing death of its E. coli host but is also indirectly involved in the lysogenic-lytic switch.

Genome-wide characterization of essential, toxicity-modulating and no-phenotype genes in S. cerevisiae

Based on the requirements for an organism's viability, genes can be classified into essential genes and non-essential genes. Non-essential genes can be further classified into toxicity-modulating genes and no-phenotype genes based on the fitness phenotype of yeast cells when the gene is deleted under DNA-damaging conditions. In this study, graph theoretical approaches were used to characterize essential, toxicity-modulating and no-phenotype genes for S. cerevisiae in the physical interaction (PI) network and the perturbation sensitivity (PS) network. We also gained previously published biological datasets to gain a more complete understanding of the differences and relationships between essential, toxicity-modulating genes and no-phenotype genes. The analysis results indicate that toxicity-modulating genes have similar properties as essential genes, and toxicity-modulating genes might represent a middle ground between essential genes and no-phenotype genes, suggesting that cells initiate highly coordinated responses to damage that are similar to those needed for vital cellular functions. These findings may elucidate the mechanisms for understanding toxicity-modulating processes relevant to certain diseases.

A naturally occurring 4-bp deletion in the intron 4 of p53 creates a spectrum of novel p53 isoforms with anti-apoptosis function

p53 functions as a tumor suppressor by transcriptionally regulating the expression of genes involved in controlling cell proliferation or apoptosis. p53 and its isoform Δ133p53/Δ113p53 form a negative regulation loop in that p53 activates the expression of Δ133p53/Δ113p53 while Δ133p53/Δ113p53 specifically antagonizes p53 apoptotic activity. This pathway is especially important to safeguard the process of embryogenesis because sudden activation of p53 by DNA damage signals or developmental stress is detrimental to a developing embryo. Here we report the identification of five novel p53 isoforms. p53β is generated due to alternative splicing of the intron 8 of p53 while the other four, namely, TA2p53, TA3p53, TA4p53 and TA5p53, result from the combination of alternative splicing of intron 1 (within intron 4 of the p53 gene) of the Δ113p53 gene and a naturally occurring CATT 4 bp deletion within the alternative splicing product in zebrafish. The CATT 4 bp deletion creates four translation start codons which are in-frame to the open reading frame of Δ113p53. We also show that TAp53 shares the same promoter with Δ113p53 and functions to antagonize p53 apoptotic activity. The identification of Δ113p53/TA2/3/4/5p53 reveals a pro-survival mechanism which operates robustly during embryogenesis in response to the DNA-damage condition.

Role of the Streptococcus mutans CRISPR-Cas systems in immunity and cell physiology.

CRISPR-Cas systems provide adaptive microbial immunity against invading viruses and plasmids. The cariogenic bacterium Streptococcus mutans UA159 has two CRISPR-Cas systems: CRISPR1 (type II-A) and CRISPR2 (type I-C) with several spacers from both CRISPR cassettes matching sequences of phage M102 or genomic sequences of other S. mutans. The deletion of the cas genes of CRISPR1 (ΔC1S), CRISPR2 (ΔC2E), or both CRISPR1+2 (ΔC1SC2E) or the removal of spacers 2 and 3 (ΔCR1SP13E) in S. mutans UA159 did not affect phage sensitivity when challenged with virulent phage M102. Using plasmid transformation experiments, we demonstrated that the CRISPR1-Cas system inhibits transformation of S. mutans by the plasmids matching the spacers 2 and 3. Functional analysis of the cas deletion mutants revealed that in addition to a role in plasmid targeting, both CRISPR systems also contribute to the regulation of bacterial physiology in S. mutans. Compared to wild-type cells, the ΔC1S strain displayed diminished growth under cell membrane and oxidative stress, enhanced growth under low pH, and had reduced survival under heat shock and DNA-damaging conditions, whereas the ΔC2E strain exhibited increased sensitivity to heat shock. Transcriptional analysis revealed that the two-component signal transduction system VicR/K differentially modulates expression of cas genes within CRISPR-Cas systems, suggesting that VicR/K might coordinate the expression of two CRISPR-Cas systems. Collectively, we provide in vivo evidence that the type II-A CRISPR-Cas system of S. mutans may be targeted to manipulate its stress response and to influence the host to control the uptake and dissemination of antibiotic resistance genes.

Repair on the go: E. coli maintains a high proliferation rate while repairing a chronic DNA double-strand break.

DNA damage checkpoints exist to promote cell survival and the faithful inheritance of genetic information. It is thought that one function of such checkpoints is to ensure that cell division does not occur before DNA damage is repaired. However, in unicellular organisms, rapid cell multiplication confers a powerful selective advantage, leading to a dilemma. Is the activation of a DNA damage checkpoint compatible with rapid cell multiplication? By uncoupling the initiation of DNA replication from cell division, the Escherichia coli cell cycle offers a solution to this dilemma. Here, we show that a DNA double-strand break, which occurs once per replication cycle, induces the SOS response. This SOS induction is needed for cell survival due to a requirement for an elevated level of expression of the RecA protein. Cell division is delayed, leading to an increase in average cell length but with no detectable consequence on mutagenesis and little effect on growth rate and viability. The increase in cell length caused by chronic DNA double-strand break repair comprises three components: two types of increase in the unit cell size, one independent of SfiA and SlmA, the other dependent of the presence of SfiA and the absence of SlmA, and a filamentation component that is dependent on the presence of either SfiA or SlmA. These results imply that chronic checkpoint induction in E. coli is compatible with rapid cell multiplication. Therefore, under conditions of chronic low-level DNA damage, the SOS checkpoint operates seamlessly in a cell cycle where the initiation of DNA replication is uncoupled from cell division.

Rad5 Plays a Major Role in the Cellular Response to DNA Damage during Chromosome Replication

The RAD6/RAD18 pathway of DNA damage tolerance overcomes unrepaired lesions that block replication forks. It is subdivided into two branches: translesion DNA synthesis, which is frequently error prone, and the error-free DNA-damage-avoidance subpathway. Here, we show that Rad5(HLTF/SHPRH), which mediates the error-free branch, has a major role in the response to DNA damage caused by methyl methanesulfonate (MMS) during chromosome replication, whereas translesion synthesis polymerases make only a minor contribution. Both the ubiquitin-ligase and the ATPase/helicase activities of Rad5 are necessary for this cellular response. We show that Rad5 is required for the progression ofreplication forks through MMS-damaged DNA. Moreover, supporting its role during replication, this protein reaches maximum levels during S phase and forms subnuclear foci when replication occurs in the presence of DNA damage. Thus, Rad5 ensures the completion of chromosome replication under DNA-damaging conditions while minimizing the risk of mutagenesis, thereby contributing significantly to genome integrity maintenance.

Abstract 1392: The Pursuit of p73 Regulation: interaction partners tell many different stories

Abstract p73 is a member of the p53 tumor suppressor family, which can trigger cell cycle arrest and apoptosis through its ability to bind p53 DNA target sites, thereby it acts as a negative regulator of tumorigenesis. Similar to p53, at normal state p73 is maintained at very low level but it rapidly gets induced upon genotoxic stress. Accumulating evidences demonstrated that p73 is mainly regulated at the protein level but its significance is still not clearly understood. It is believed that it is a consequence of post-translational modifications (PTMs) of p73 upon DNA damage. Emerging reports suggested that other than PTMs, various co-factors are responsible for regulating its level and activity. But all these reports remained inadequate to explain the molecular mechanisms behind regulation and activity of p73 under non-stressed and stressed conditions. The aim of our study is to find out novel interactors of p73 which distinctively affect its stability and activity in normal state and upon DNA damage. We employed a proteomic approach to identify differential interactors of p73 under unstressed and genotoxic stress conditions. We prepared adenovirus system to ectopically overexpress dual-tagged p73 into cells. Our tandem affinity purification screening lead us to identification of many p73-associated proteins. Among the novel interactors, TRIM28, a RING-type E3 ligase, was of particular interest because it interacts with p73 under unstressed condition but no visible interaction was detected after DNA damage. We show here that TRIM28 ubiquitylates and targets p73 for proteasome-mediated degradation in unstressed conditions. Upon genotoxic stress, p73 gets phosphorylated at tyrosine 99 position in c-Abl-dependent manner leading to abrogation of its interaction with TRIM28 which subsequently leads to p73 stabilization. Another protein, MED15 was of particular interest because it interacts with p73 in phosphorylation-dependent manner and it serves as a coactivator of p73. Interestingly, we found that phosphorylation of p73 is a prerequisite for its interaction with MED15. Furthermore, loss of MED15 leads to decline in p73-mediated transactivation of its downstream target genes under DNA damaging conditions. Therefore, we propose a model in which DNA damage-induced phosphorylation of p73 affects its interaction with its negative regulator and leads to its preferential association with its coactivator. Citation Format: Yatendra K. Satija, Sanjeev Das. The Pursuit of p73 Regulation: interaction partners tell many different stories. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1392. doi:10.1158/1538-7445.AM2014-1392