A new role for Escherichia coli Dam DNA methylase in prevention of aberrant chromosomal replication.

The Dam DNA methylase of Escherichia coli is required for methyl-directed mismatch repair, regulation of chromosomal DNA replication initiation from oriC (which is DnaA-dependent), and regulation of gene expression. Here, we show that Dam suppresses aberrant oriC-independent chromosomal replication (also called constitutive stable DNA replication, or cSDR). Dam deficiency conferred cSDR and, in presence of additional mutations (Δtus, rpoB*35) that facilitate retrograde replication fork progression, rescued the lethality of ΔdnaA mutants. The DinG helicase was required for rescue of ΔdnaA inviability during cSDR. Viability of ΔdnaA dam derivatives was dependent on the mismatch repair proteins, since such viability was lost upon introduction of deletions in mutS, mutH or mutL; thus generation of double strand ends (DSEs) by MutHLS action appears to be required for cSDR in the dam mutant. On the other hand, another DSE-generating agent phleomycin was unable to rescue ΔdnaA lethality in dam+ derivatives (mutS+ or ΔmutS), but it could do so in the dam ΔmutS strain. These results point to a second role for Dam deficiency in cSDR. We propose that in Dam-deficient strains, there is an increased likelihood of reverse replication restart (towards oriC) following recombinational repair of DSEs on the chromosome.

Fork pausing allows centromere DNA loop formation and kinetochore assembly.

De novo kinetochore assembly, but not template-directed assembly, is dependent on COMA, the kinetochore complex engaged in cohesin recruitment. The slowing of replication fork progression by treatment with phleomycin (PHL), hydroxyurea, or deletion of the replication fork protection protein Csm3 can activate de novo kinetochore assembly in COMA mutants. Centromere DNA looping at the site of de novo kinetochore assembly can be detected shortly after exposure to PHL. Using simulations to explore the thermodynamics of DNA loops, we propose that loop formation is disfavored during bidirectional replication fork migration. One function of replication fork stalling upon encounters with DNA damage or other blockades may be to allow time for thermal fluctuations of the DNA chain to explore numerous configurations. Biasing thermodynamics provides a mechanism to facilitate macromolecular assembly, DNA repair, and other nucleic acid transactions at the replication fork. These loop configurations are essential for sister centromere separation and kinetochore assembly in the absence of the COMA complex.

Structurally distinct Mre11 domains mediate MRX functions in resection, end-tethering and DNA damage resistance.

Sae2 cooperates with the Mre11-Rad50-Xrs2 (MRX) complex to initiate resection of DNA double-strand breaks (DSBs) and to maintain the DSB ends in close proximity to allow their repair. How these diverse MRX-Sae2 functions contribute to DNA damage resistance is not known. Here, we describe mre11 alleles that suppress the hypersensitivity of sae2Δ cells to genotoxic agents. By assessing the impact of these mutations at the cellular and structural levels, we found that all the mre11 alleles that restore sae2Δ resistance to both camptothecin and phleomycin affect the Mre11 N-terminus and suppress the resection defect of sae2Δ cells by lowering MRX and Tel1 association to DSBs. As a consequence, the diminished Tel1 persistence potentiates Sgs1-Dna2 resection activity by decreasing Rad9 association to DSBs. By contrast, the mre11 mutations restoring sae2Δ resistance only to phleomycin are located in Mre11 C-terminus and bypass Sae2 function in end-tethering but not in DSB resection, possibly by destabilizing the Mre11-Rad50 open conformation. These findings unmask the existence of structurally distinct Mre11 domains that support resistance to genotoxic agents by mediating different processes.

DksA and DNA double-strand break repair.

We use genetic assays to suggest that transcription-coupled repair or new origin formation in Escherichia coli involves removal of RNAP to create an RNA primer for DNA synthesis. Transcription factor DksA was shown to play a role in numerous reactions involving RNA polymerase. Some, but not all, of the activities of DksA at promoters or during transcription elongation require (p)ppGpp. In addition to its role during transcription, DksA is also involved in maintaining genome integrity. Cells lacking DksA are sensitive to multiple DNA damaging agents including UV light, ionizing radiation, mitomycin C, and nalidixic acid. Here, we focus on two recent studies addressing the importance of DksA in the repair of double-strand breaks (DSBs), one by Sivaramakrishnan et al. (Nature 550:214-218, 2017) and one originating in our laboratory, Myka et al. (Mol Microbiol 111:1382-1397. https://doi.org/10.1111/mmi.14227 , 2019). It appears that depending on the type and possibly location of DNA damage, DksA can play either a passive or an active role in DSB repair. The passive role relies on exclusion of anti-backtracking factors from the RNAP secondary channel. The exact mechanism of active DksA-mediated DNA repair is unknown. However, DksA was proposed to destabilize transcription complexes, thus clearing the way for recombination and DNA repair. Based on the requirement for DksA, both in repair of DSBs and the R-loop-dependent formation of new origins of DNA replication, we propose that DksA may allow for removal of RNAP without unwinding of the RNA:DNA hybrid, which can then be extended by a DNA polymerase. This mechanism obviates the need for RNAP backtracking to repair damaged DNA.

Rgf1p (Rho1p GEF) is required for double-strand break repair in fission yeast.

Rho GTPases are conserved molecules that control cytoskeletal dynamics. These functions are expedited by Rho GEFs that stimulate the release of GDP to enable GTP binding, thereby allowing Rho proteins to initiate intracellular signaling. How Rho GEFs and Rho GTPases protect cells from DNA damage is unknown. Here, we explore the extreme sensitivity of a deletion mutation in the Rho1p exchange factor Rgf1p to the DNA break/inducing antibiotic phleomycin (Phl). The Rgf1p mutant cells are defective in reentry into the cell cycle following the induction of severe DNA damage. This phenotype correlates with the inability of rgf1Δ cells to efficiently repair fragmented chromosomes after Phl treatment. Consistent with this observation Rad11p (ssDNA binding protein, RPA), Rad52p, Rad54p and Rad51p, which facilitate strand invasion in the process of homology-directed repair (HDR), are permanently stacked in Phl-induced foci in rgf1Δ cells. These phenotypes are phenocopied by genetic inhibition of Rho1p. Our data provide evidence that Rgf1p/Rho1p activity positively controls a repair function that confers resistance against the anti-cancer drug Phl.

Polymerases ε and ∂ repair dysfunctional telomeres facilitated by salt.

Damaged DNA can be repaired by removal and re-synthesis of up to 30 nucleotides during base or nucleotide excision repair. An important question is what happens when many more nucleotides are removed, resulting in long single-stranded DNA (ssDNA) lesions. Such lesions appear on chromosomes during telomere damage, double strand break repair or after the UV damage of stationary phase cells. Here, we show that long single-stranded lesions, formed at dysfunctional telomeres in budding yeast, are re-synthesized when cells are removed from the telomere-damaging environment. This process requires Pol32, an accessory factor of Polymerase δ. However, re-synthesis takes place even when the telomere-damaging conditions persist, in which case the accessory factors of both polymerases δ and ε are required, and surprisingly, salt. Salt added to the medium facilitates the DNA synthesis, independently of the osmotic stress responses. These results provide unexpected insights into the DNA metabolism and challenge the current view on cellular responses to telomere dysfunction.

Physical Association of Saccharomyces cerevisiae Polo-like Kinase Cdc5 with Chromosomal Cohesin Facilitates DNA Damage Response.

At the onset of anaphase, a protease called separase breaks the link between sister chromatids by cleaving the cohesin subunit Scc1. This irreversible step in the cell cycle is promoted by degradation of the separase inhibitor, securin, and polo-like kinase (Plk) 1-dependent phosphorylation of the Scc1 subunit. Plk could recognize substrates through interaction between its phosphopeptide interaction domain, the polo-box domain, and a phosphorylated priming site in the substrate, which has been generated by a priming kinase beforehand. However, the physiological relevance of this targeting mechanism remains to be addressed for many of the Plk1 substrates. Here, we show that budding yeast Plk1, Cdc5, is pre-deposited onto cohesin engaged in cohesion on chromosome arms in G2/M phase cells. The Cdc5-cohesin association is mediated by direct interaction between the polo-box domain of Cdc5 and Scc1 phosphorylated at multiple sites in its middle region. Alanine substitutions of the possible priming phosphorylation sites (scc1-15A) impair Cdc5 association with chromosomal cohesin, but they make only a moderate impact on mitotic cell growth even in securin-deleted cells (pds1Δ), where Scc1 phosphorylation by Cdc5 is indispensable. The same scc1-15A pds1Δ double mutant, however, exhibits marked sensitivity to the DNA-damaging agent phleomycin, suggesting that the priming phosphorylation of Scc1 poses an additional layer of regulation that enables yeast cells to adapt to genotoxic environments.

Development of a sufficient and effective procedure for transformation of an oleaginous yeast, Rhodosporidium toruloides DMKU3-TK16.

Rhodosporidium toruloides DMKU3-TK16 (TK16), a basidiomycetous yeast isolated in Thailand, can produce a large amount of oil corresponding to approximately 70 % of its dry cell weight. However, lack of a sufficient and efficient transformation method makes further genetic manipulation of this organism difficult. We here developed a new transformation system for R. toruloides using a lithium acetate method with the Sh ble gene as a selective marker under the control of the R. toruloides ATCC 10657 GPD1 promoter. A linear DNA fragment containing the Sh ble gene expression cassette was integrated into the genome, and its integration was confirmed by colony PCR and Southern blot. Then, we further optimized the parameters affecting the transformation efficiency, such as the amount of linear DNA, the growth phase, the incubation time in the transformation mixture, the heat shock treatment temperature, the addition of DMSO and carrier DNA, and the recovery incubation time. With the developed method, the transformation efficiency of approximately 25 transformants/µg DNA was achieved. Compared with the initial trial, transformation efficiency was enhanced 417-fold. We further demonstrated the heterologous production of EGFP in TK16 by microscopic observation and immunoblot analysis, and use the technique to disrupt the endogenous URA3 gene. The newly developed method is thus simple and time saving, making it useful for efficient introduction of an exogenous gene into R. toruloides strains. Accordingly, this new practical approach should facilitate the molecular manipulation, such as target gene introduction and deletion, of TK16 and other R. toruloides strains as a major source of biodiesel.

Variable NF-κB pathway responses in colon cancer cells treated with chemotherapeutic drugs.

BACKGROUND: The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway is activated in cells exposed to various stimuli, including those originating on the cell surface or in the nucleus. Activated NF-κB signaling is thought to enhance cell survival in response to these stimuli, which include chemotherapy and radiation. In the present effort, we determined which anticancer drugs preferentially activate NF-κB in colon cancer cells. METHODS: NF-κB reporter cells were established and treated with 5-fluorouracil (5-FU, DNA/RNA damaging), oxaliplatin (DNA damaging), camptothecin (CTP, topoisomerase inhibitor), phleomycin (radiomimetic), or erlotinib (EGFR inhibitor). The activation of NF-κB was assessed by immunofluorescence for p65 translocation, luciferase assays, and downstream targets of NF-κB activation (cIAP2, and Bcl-XL) were evaluated by immunoblotting, by ELISA (CXCL8 and IL-6 in culture supernatants), or by gene expression analysis. RESULTS: Colon cancer cells responded variably to different classes of therapeutic agents, and these agents initiated variable responses among different cell types. CPT activated NF-κB in SW480 colon cancer cells in a dose-dependent manner, but not in HCT116 cells that were either wild-type or deficient for p53. In SW480 colon cancer cells, NF-κB activation by CPT was accompanied by secretion of the cytokine CXCL8, but not by up-regulation of the anti-apoptotic genes, cIAP2 or Bcl-XL. On the contrary, treatment of HCT116 cells with CPT resulted in up-regulation of CXCR2, a receptor for CXCL8, without an increase in cytokine levels. In SW480 cells, NF-κB reporter activity, but not cytokine secretion, was inhibited by SM-7368, an NF-κB inhibitor. CONCLUSION: The results show that, in response to cancer therapeutic agents, NF-κB activation varies with the cellular make up and that drug-induced NF-κB activation may be functionally uncoupled from anti-apoptotic outcomes found for other stimuli. Some cancer cells in a heterogeneous tumor tissue may, under therapeutic pressure, release soluble factors that have paracrine activity on neighboring cells that express the cognate receptors.

Transformation of Candida guilliermondii wild-type strains using the Staphylococcus aureus MRSA 252 ble gene as a phleomycin-resistant marker.

We designed an efficient transformation system for Candida guilliermondii wild-type strains. We demonstrated that the Staphylococcus aureus MRSA 252 ble coding sequence placed under the control of the yeast phosphoglycerate kinase gene transcription-regulating regions confers phleomycin resistance to transformed C. guilliermondii cells. To illustrate the potential of this drug-resistant cassette, we carried out the disruption of the C. guilliermondii ADE2 gene. This new dominant selectable marker represents a powerful tool to study the function of various genes in this yeast of clinical and biotechnological interest.

Dual gene expression cassette vectors with antibiotic selection markers for engineering in Saccharomyces cerevisiae.

BACKGROUND: Manipulations in Saccharomyces cerevisiae classically depend on use of auxotrophy selection markers. There are several disadvantages to this in a microbial cell factory setting: (1) auxotrophies must first be engineered in prototrophic strains, and many industrial strains are polyploid/aneuploid prototrophs (2) available strain auxotrophies must be paired with available repair plasmids (3) remaining auxotrophies must be repaired prior to development of industrial bioprocesses. Use of dominant antibiotic resistance markers can circumvent these problems. However, there are relatively few yeast antibiotic resistance marker vectors available; furthermore, available vectors contain only one expression cassette, and it is often desirable to introduce more than one gene at a time. RESULTS: To overcome these problems, eight new shuttle vectors have been developed. The plasmids are maintained in yeast under a 2 µm ori and in E. coli by a pUC ori. They contain two yeast expression cassettes driven by either (1) the constitutive TEF1 and PGK1 promoters, or (2) the constitutive TEF1 promoter and the inducible GAL10 or HXT7 promoters. Expression strength of these promoters over a typical production time frame in glucose/galactose medium was examined, and identified the TEF1 and HXT7 promoters as preferred promoters over long term fermentations. Selection is provided by either aphA1 (conferring resistance to G418 in yeast and kanamycin/neomycin in E. coli) or ble (conferring resistance to phleomycin in both yeast and E. coli). Selection conditions for these plasmids/antibiotics in defined media were examined, and selection considerations are reviewed. In particular, medium pH has a strong effect on both G418 and phleomycin selection. CONCLUSIONS: These vectors allow manipulations in prototrophic yeast strains with expression of two gene cassettes per plasmid, and will be particularly useful for metabolic engineering applications. The vector set expands the (currently limited) selection of antibiotic marker plasmids available for use in yeast, and in addition makes available dual gene expression cassettes on individual plasmids using antibiotic selection. The resistance gene cassettes are flanked by loxP recognition sites to allow CreA-mediated marker removal and recycling, providing the potential for genomic integration of multiple genes. Guidelines for selection using G418 and phleomycin are provided.

The natural absence of RPA1N domain did not impair Leishmania amazonensis RPA-1 participation in DNA damage response and telomere protection.

We have previously shown that the subunit 1 of Leishmania amazonensis RPA (LaRPA-1) alone binds the G-rich telomeric strand and is structurally different from other RPA-1. It is analogous to telomere end-binding proteins described in model eukaryotes whose homologues were not identified in the protozoan´s genome. Here we show that LaRPA-1 is involved with damage response and telomere protection although it lacks the RPA1N domain involved with the binding with multiple checkpoint proteins. We induced DNA double-strand breaks (DSBs) in Leishmania using phleomycin. Damage was confirmed by TUNEL-positive nuclei and triggered a G1/S cell cycle arrest that was accompanied by nuclear accumulation of LaRPA-1 and RAD51 in the S phase of hydroxyurea-synchronized parasites. DSBs also increased the levels of RAD51 in non-synchronized parasites and of LaRPA-1 and RAD51 in the S phase of synchronized cells. More LaRPA-1 appeared immunoprecipitating telomeres in vivo and associated in a complex containing RAD51, although this interaction needs more investigation. RAD51 apparently co-localized with few telomeric clusters but it did not immunoprecipitate telomeric DNA. These findings suggest that LaRPA-1 and RAD51 work together in response to DNA DSBs and at telomeres, upon damage, LaRPA-1 works probably to prevent loss of single-stranded DNA and to assume a capping function.

Loss of function of FIGNL1, a DNA damage response gene, causes human ovarian dysgenesis.

Ovarian dysgenesis (OD), an XX disorder of sex development, presents with primary amenorrhea, hypergonadotrophic hypogonadism, and infertility. In an Ashkenazi Jewish patient with OD, whole exome sequencing identified compound heterozygous frameshifts in FIGNL1, a DNA damage response (DDR) gene: c.189del and c.1519_1523del. Chromosomal breakage was significantly increased in patient cells, both spontaneously, and following mitomycin C exposure. Transfection of DYK-tagged FIGNL1 constructs in HEK293 cells showed no detectable protein in FIGNL1c.189del and truncation with reduced expression in FIGNL1c.1519_1523del (64% of wild-type [WT], P = .003). FIGNL1 forms nuclear foci increased by phleomycin treatment (20.6 ± 1.6 vs 14.8 ± 2.4, P = .02). However, mutant constructs showed reduced DYK-FIGNL1 foci formation in non-treated cells (0.8 ± 0.9 and 5.6 ± 1.5 vs 14.8 ± 2.4 in DYK-FIGNL1WT, P < .001) and no increase with phleomycin treatment. In conclusion, FIGNL1 loss of function is a newly characterized OD gene, highlighting the DDR pathway's role in ovarian development and maintenance and suggesting chromosomal breakage as an assessment tool in XX-DSD patients.

Deciphering the mechanism of processive ssDNA digestion by the Dna2-RPA ensemble.

Single-stranded DNA (ssDNA) commonly occurs as intermediates in DNA metabolic pathways. The ssDNA binding protein, RPA, not only protects the integrity of ssDNA, but also directs the downstream factor that signals or repairs the ssDNA intermediate. However, it remains unclear how these enzymes/factors outcompete RPA to access ssDNA. Using the budding yeast Saccharomyces cerevisiae as a model system, we find that Dna2 - a key nuclease in DNA replication and repair - employs a bimodal interface to act with RPA both in cis and in trans. The cis-activity makes RPA a processive unit for Dna2-catalyzed ssDNA digestion, where RPA delivers its bound ssDNA to Dna2. On the other hand, activity in trans is mediated by an acidic patch on Dna2, which enables it to function with a sub-optimal amount of RPA, or to overcome DNA secondary structures. The trans-activity mode is not required for cell viability, but is necessary for effective double strand break (DSB) repair.

A GFP-based assay to quantify the impact of effectors on the ex planta development of the slowly growing barley pathogen Rhynchosporium commune.

A growth assay was established for the barley pathogen Rhynchosporium commune with EGFP-tagged fungal mutants. This assay was used to study the effect of four antibiotics (hygromycin B, nourseothricin, kanamycin, phleomycin) and a herbicide (phosphinothricin) on fungal development. Fitting the growth curves to the modified Gompertz model allowed calculating growth parameters, such as lag periods of fungal colony formation and mycelial growth rates as well as EC(50) values. Except kanamycin all compounds were efficient inhibitors so that the corresponding resistance-conferring genes can be used as markers for selection of fungal transformation-based mutants. In addition the assay was used to quantify the inhibitory activity of a barley secondary metabolite, the indole alkaloid gramine.

DNA-damage response in the basidiomycete fungus Ustilago maydis relies in a sole Chk1-like kinase.

Chk1 is a protein kinase that acts as a key signal transducer within the complex network responsible of the cellular response to different DNA damages. It is a conserved element along the eukaryotic kingdom, together with a second checkpoint kinase, called Chk2/Rad53. In fact, all organisms studied so far carried at least one copy of each kind of checkpoint kinase. Since the relative contribution to the DNA-damage response of each type of kinase varies from one organism to other, the current view about the roles of Chk1 and Chk2/Rad53 during DNA-damage response is one of mutual complementation and intimate cooperation. However, in this work it is reported that Ustilago maydis - a phytopathogenic fungus exhibiting extreme resistance to UV and ionizing radiation - have a single kinase belonging to the Chk1 family but strikingly no kinases related to Chk2/Rad53 family are apparent. The U. maydis Chk1 kinase is able to respond to different classes of DNA damages and its activity is required for the cellular adaptation to such damages. As other described components of the Chk1 family of kinases, U. maydis Chk1 is phosphorylated and translocated to nucleus in response to DNA-damage signals. Interestingly subtle differences in this response depending on the kind of DNA damage are apparent, suggesting that in U. maydis the sole Chk1 kinase recapitulates the roles that in other organisms are shared by Chk1 and the Chk2/Rad53 family of protein kinases.

Bystander effects induced by chemicals and ionizing radiation: evaluation of changes in gene expression of downstream MAPK targets.

Radiation-induced bystander effects have been evaluated extensively, including the involvement of the mitogen-activated protein kinase (MAPK) pathways. However, few studies have examined the ability of chemicals to induce bystander effects, and the molecular mechanisms involved in chemical bystander effects have not been investigated. We have previously demonstrated the ability of mitomycin C (MMC) and phleomycin (PHL) to induce bystander effects in normal human lymphoblastoid cells. Here, we demonstrate changes in the expression of MAPK target genes following bystander exposure to MMC or PHL or ionizing radiation. The expression changes of 18 genes, which code for proteins that are downstream targets of MAPK proteins, were evaluated at various time points following direct or bystander exposure to MMC, PHL and ionizing radiation. The 18 genes were analysed as groups belonging to one of the seven possible combinations of the three MAPK pathways. We observed statistically significant changes in expression of several genes following exposure to each agent. However, when the expression changes were analysed in the bystander cells alone, significant increases in expression of MAPK target genes were observed for MMC- and radiation-induced bystander effects but not for PHL. PHL is an acknowledged radiomimetic agent; however, in the present study, PHL responses did not resemble those of radiation. These results provide evidence for bystander-induced changes in MAPK proteins and downstream targets and suggest that the bystander effects are a part of a general stress response.

Several tetratricopeptide repeat (TPR) motifs of FANCG are required for assembly of the BRCA2/D1-D2-G-X3 complex, FANCD2 monoubiquitylation and phleomycin resistance.

The Fanconi anaemia (FA) FANCG protein is an integral component of the FA nuclear core complex that is required for monoubiquitylation of FANCD2. FANCG is also part of another protein complex termed D1-D2-G-X3 that contains FANCD2 and the homologous recombination repair proteins BRCA2 (FANCD1) and XRCC3. Formation of the D1-D2-G-X3 complex is mediated by serine-7 phosphorylation of FANCG and occurs independently of the FA core complex and FANCD2 monoubiquitylation. FANCG contains seven tetratricopeptide repeat (TPR) motifs that mediate protein-protein interactions and here we show that mutation of several of the TPR motifs at a conserved consensus residue ablates the in vivo binding activity of FANCG. Expression of mutated TPR1, TPR2, TPR5 and TPR6 in Chinese hamster fancg mutant NM3 fails to functionally complement its hypersensitivities to mitomycin C (MMC) and phleomycin and fails to restore FANCD2 monoubiquitylation. Using co-immunoprecipitation analysis, we demonstrate that these TPR-mutated FANCG proteins fail to interact with BRCA2, XRCC3, FANCA or FANCF. The interactions of other proteins in the D1-D2-G-X3 complex are also absent, including the interaction of BRCA2 with both the monoubiquitylated (FANCD2-L) and non-ubiquitylated (FANCD2-S) isoforms of FANCD2. Interestingly, a mutation of TPR7 (R563E), that complements the MMC and phleomycin hypersensitivity of human FA-G EUFA316 cells, fails to complement NM3, despite the mutated FANCG protein co-precipitating with FANCA, BRCA2 and XRCC3. Whilst interaction of TPR7-mutated FANCG with FANCF does appear to be reduced in NM3, FANCD2 is monoubiquitylated suggesting that sub-optimal interactions of FANCG in the core complex and the D1-D2-G-X3 complex are responsible for the observed MMC- and phleomycin-hypersensitivity, rather than a defect in FANCD2 monoubiquitylation. Our data demonstrate that FANCG functions as a mediator of protein-protein interactions and is vital for the assembly of multi-protein complexes including the FA core complex and the D1-D2-G-X3 complex.

Involvement of MAPK proteins in bystander effects induced by chemicals and ionizing radiation.

Many studies have examined bystander effects induced by ionizing radiation, however few have evaluated the ability of chemicals to induce similar effects. We previously reported the ability of two chemicals, mitomycin C (MMC) and phleomycin (PHL) to induce bystander effects in normal human lymphoblastoid cell lines. The focus of the current study was to determine the involvement of the MAPK proteins in bystander effects induced by physical and chemical DNA damaging agents and to evaluate the effects of MAPK inhibition on bystander-induced caspase 3/7 activation. The phosphorylation levels of the MAPK proteins ERK1/2, JNK, and p38, were measured from 1 to 24h following direct or bystander exposure to MMC, PHL or radiation. We observed transient phosphorylation, at early time points, of all 3 proteins in bystander cells. We also evaluated the effect of MAPK inhibition on bystander-induced caspase 3/7 activity to determine the role of MAPK proteins in bystander-induced apoptosis. We observed bystander-induced activation of caspase 3/7 in bystander cells. Inhibition of MAPK proteins resulted in a decrease in caspase 3/7 activity at the early time points, and the caspase activity increased (in the case of ERK inhibition) or returned to basal levels (in the case of JNK or p38 inhibition) between 12 and 24h. PHL is considered to be a radiomimetic agent, however in the present study PHL behaved more like a chemical and not like radiation in terms of MAPK phosphorylation. These results point to the involvement of MAPK proteins in the bystander effect induced by radiation and chemicals and provide additional evidence that this response is not limited to radiation but is a generalized stress response in cells.

Inositol pyrophosphates modulate hydrogen peroxide signalling.

Inositol pyrophosphates are involved in a variety of cellular functions, but the specific pathways and/or downstream targets remain poorly characterized. In the present study we use Saccharomyces cerevisiae mutants to examine the potential roles of inositol pyrophosphates in responding to cell damage caused by ROS (reactive oxygen species). Yeast lacking kcs1 [the S. cerevisiae IP6K (inositol hexakisphosphate kinase)] have greatly reduced IP7 (diphosphoinositol pentakisphosphate) and IP8 (bisdiphosphoinositol tetrakisphosphate) levels, and display increased resistance to cell death caused by H2O2, consistent with a sustained activation of DNA repair mechanisms controlled by the Rad53 pathway. Other Rad53-controlled functions, such as actin polymerization, appear unaffected by inositol pyrophosphates. Yeast lacking vip1 [the S. cerevisiae PP-IP5K (also known as IP7K, IP7 kinase)] accumulate large amounts of the inositol pyrophosphate IP7, but have no detectable IP8, indicating that this enzyme represents the physiological IP7 kinase. Similar to kcs1Delta yeast, vip1Delta cells showed an increased resistance to cell death caused by H2O2, indicating that it is probably the double-pyrophosphorylated form of IP8 [(PP)2-IP4] which mediates the H2O2 response. However, these inositol pyrophosphates are not involved in directly sensing DNA damage, as kcs1Delta cells are more responsive to DNA damage caused by phleomycin. We observe in vivo a rapid decrease in cellular inositol pyrophosphate levels following exposure to H2O2, and an inhibitory effect of H2O2 on the enzymatic activity of Kcs1 in vitro. Furthermore, parallel cysteine mutagenesis studies performed on mammalian IP6K1 are suggestive that the ROS signal might be transduced by the direct modification of this evolutionarily conserved class of enzymes.