Targeted nuclear irradiation with a proton microbeam induces oxidative DNA base damage and triggers the recruitment of DNA glycosylases OGG1 and NTH1.

DNA is the major target of radiation therapy of malignant tumors. Ionizing radiation (IR) induces a variety of DNA lesions, including chemically modified bases and strand breaks. The use of proton beam therapy for cancer treatment is ramping up, as it is expected to reduce normal tissue damage. Thus, it is important to understand the molecular mechanisms of recognition, signaling, and repair of DNA damage induced by protons in the perspective of assessing not only the risk associated with human exposure to IR but also the possibility to improve the efficacy of therapy. Here, we used targeted irradiation of nuclear regions of living cells with controlled number of protons at a high spatio-temporal resolution to detect the induced base lesions and characterize the recruitment kinetics of the specific DNA glycosylases to DNA damage sites. We show that localized irradiation with 4 MeV protons induces, in addition to DNA double strand breaks (DSBs), the oxidized bases 7,8-dihydro-8-oxoguanine (8-oxoG) and thymine glycol (TG) at the site of irradiation. Consistently, the DNA glycosylases OGG1 and NTH1, capable of excising 8-oxoG and TG, respectively, and initiating the base excision repair (BER) pathway, are recruited to the site of damage. To our knowledge, this is the first direct evidence indicating that proton microbeams induce oxidative base damage, and thus implicating BER in the repair of DNA lesions induced by protons.

Identification of key residues of the DNA glycosylase OGG1 controlling efficient DNA sampling and recruitment to oxidized bases in living cells.

The DNA-glycosylase OGG1 oversees the detection and clearance of the 7,8-dihydro-8-oxoguanine (8-oxoG), which is the most frequent form of oxidized base in the genome. This lesion is deeply buried within the double-helix and its detection requires careful inspection of the bases by OGG1 via a mechanism that remains only partially understood. By analyzing OGG1 dynamics in the nucleus of living human cells, we demonstrate that the glycosylase constantly samples the DNA by rapidly alternating between diffusion within the nucleoplasm and short transits on the DNA. This sampling process, that we find to be tightly regulated by the conserved residue G245, is crucial for the rapid recruitment of OGG1 at oxidative lesions induced by laser micro-irradiation. Furthermore, we show that residues Y203, N149 and N150, while being all involved in early stages of 8-oxoG probing by OGG1 based on previous structural data, differentially regulate the sampling of the DNA and recruitment to oxidative lesions.

A noncanonical response to replication stress protects genome stability through ROS production, in an adaptive manner.

Cells are inevitably challenged by low-level/endogenous stresses that do not arrest DNA replication. Here, in human primary cells, we discovered and characterized a noncanonical cellular response that is specific to nonblocking replication stress. Although this response generates reactive oxygen species (ROS), it induces a program that prevents the accumulation of premutagenic 8-oxoguanine in an adaptive way. Indeed, replication stress-induced ROS (RIR) activate FOXO1-controlled detoxification genes such as SEPP1, catalase, GPX1, and SOD2. Primary cells tightly control the production of RIR: They are excluded from the nucleus and are produced by the cellular NADPH oxidases DUOX1/DUOX2, whose expression is controlled by NF-κB, which is activated by PARP1 upon replication stress. In parallel, inflammatory cytokine gene expression is induced through the NF-κB-PARP1 axis upon nonblocking replication stress. Increasing replication stress intensity accumulates DNA double-strand breaks and triggers the suppression of RIR by p53 and ATM. These data underline the fine-tuning of the cellular response to stress that protects genome stability maintenance, showing that primary cells adapt their responses to replication stress severity.

OGG1 competitive inhibitors show important off-target effects by directly inhibiting efflux pumps and disturbing mitotic progression.

One of the most abundant DNA lesions induced by Reactive oxygen species (ROS) is 8-oxoG, a highly mutagenic lesion that compromises genetic instability when not efficiently repaired. 8-oxoG is specifically recognized by the DNA-glycosylase OGG1 that excises the base and initiates the Base Excision Repair pathway (BER). Furthermore, OGG1 has not only a major role in DNA repair but it is also involved in transcriptional regulation. Cancer cells are particularly exposed to ROS, thus challenging their capacity to process oxidative DNA damage has been proposed as a promising therapeutic strategy for cancer treatment. Two competitive inhibitors of OGG1 (OGG1i) have been identified, TH5487 and SU0268, which bind to the OGG1 catalytic pocket preventing its fixation to the DNA. Early studies with these inhibitors show an enhanced cellular sensitivity to cytotoxic drugs and a reduction in the inflammatory response. Our study uncovers two unreported off-targets effects of these OGG1i that are independent of OGG1. In vitro and in cellulo approaches have unveiled that OGG1i TH5487 and SU0268, despite an unrelated molecular structure, are able to inhibit some members of the ABC family transporters, in particular ABC B1 (MDR1) and ABC G2 (BCRP). The inhibition of these efflux pumps by OGG1 inhibitors results in a higher intra-cellular accumulation of various fluorescent probes and drugs, and largely contributes to the enhanced cytotoxicity observed when the inhibitors are combined with cytotoxic agents. Furthermore, we found that SU0268 has an OGG1-independent anti-mitotic activity-by interfering with metaphase completion-resulting in a high cellular toxicity. These two off-target activities are observed at concentrations of OGG1i that are normally used for in vivo studies. It is thus critical to consider these previously unreported non-specific effects when interpreting studies using TH5487 and SU0268 in the context of OGG1 inhibition. Additionally, our work highlights the persistent need for new specific inhibitors of the enzymatic activity of OGG1.

ComFC mediates transport and handling of single-stranded DNA during natural transformation.

The ComFC protein is essential for natural transformation, a process that plays a major role in the spread of antibiotic resistance genes and virulence factors across bacteria. However, its role remains largely unknown. Here, we show that Helicobacter pylori ComFC is involved in DNA transport through the cell membrane, and is required for the handling of the single-stranded DNA once it is delivered into the cytoplasm. The crystal structure of ComFC includes a zinc-finger motif and a putative phosphoribosyl transferase domain, both necessary for the protein's in vivo activity. Furthermore, we show that ComFC is a membrane-associated protein with affinity for single-stranded DNA. Our results suggest that ComFC provides the link between the transport of the transforming DNA into the cytoplasm and its handling by the recombination machinery.

Tumor resistance to radiotherapy is triggered by an ATM/TAK1-dependent-increased expression of the cellular prion protein.

In solid cancers, high expression of the cellular prion protein (PrPC) is associated with stemness, invasiveness, and resistance to chemotherapy, but the role of PrPC in tumor response to radiotherapy is unknown. Here, we show that, in neuroblastoma, breast, and colorectal cancer cell lines, PrPC expression is increased after ionizing radiation (IR) and that PrPC deficiency increases radiation sensitivity and decreases radiation-induced radioresistance in tumor cells. In neuroblastoma cells, IR activates ATM that triggers TAK1-dependent phosphorylation of JNK and subsequent activation of the AP-1 transcription factor that ultimately increases PRNP promoter transcriptional activity through an AP-1 binding site in the PRNP promoter. Importantly, we show that this ATM-TAK1-PrPC pathway mediated radioresistance is activated in all tumor cell lines studied and that pharmacological inhibition of TAK1 activity recapitulates the effects of PrPC deficiency. Altogether, these results unveil how tumor cells activate PRNP to acquire resistance to radiotherapy and might have implications for therapeutic targeting of solid tumors radioresistance.

A peptide of a type I toxin-antitoxin system induces Helicobacter pylori morphological transformation from spiral shape to coccoids.

Toxin-antitoxin systems are found in many bacterial chromosomes and plasmids with roles ranging from plasmid stabilization to biofilm formation and persistence. In these systems, the expression/activity of the toxin is counteracted by an antitoxin, which, in type I systems, is an antisense RNA. While the regulatory mechanisms of these systems are mostly well defined, the toxins' biological activity and expression conditions are less understood. Here, these questions were investigated for a type I toxin-antitoxin system (AapA1-IsoA1) expressed from the chromosome of the human pathogen Helicobacter pylori We show that expression of the AapA1 toxin in H. pylori causes growth arrest associated with rapid morphological transformation from spiral-shaped bacteria to round coccoid cells. Coccoids are observed in patients and during in vitro growth as a response to different stress conditions. The AapA1 toxin, first molecular effector of coccoids to be identified, targets H. pylori inner membrane without disrupting it, as visualized by cryoelectron microscopy. The peptidoglycan composition of coccoids is modified with respect to spiral bacteria. No major changes in membrane potential or adenosine 5'-triphosphate (ATP) concentration result from AapA1 expression, suggesting coccoid viability. Single-cell live microscopy tracking the shape conversion suggests a possible association of this process with cell elongation/division interference. Oxidative stress induces coccoid formation and is associated with repression of the antitoxin promoter and enhanced processing of its transcript, leading to an imbalance in favor of AapA1 toxin expression. Our data support the hypothesis of viable coccoids with characteristics of dormant bacteria that might be important in H. pylori infections refractory to treatment.

Chromatin recruitment of OGG1 requires cohesin and mediator and is essential for efficient 8-oxoG removal.

One of the most abundant DNA lesions induced by oxidative stress is the highly mutagenic 8-oxoguanine (8-oxoG), which is specifically recognized by 8-oxoguanine DNA glycosylase 1 (OGG1) to initiate its repair. How DNA glycosylases find small non-helix-distorting DNA lesions amongst millions of bases packaged in the chromatin-based architecture of the genome remains an open question. Here, we used a high-throughput siRNA screening to identify factors involved in the recognition of 8-oxoG by OGG1. We show that cohesin and mediator subunits are required for re-localization of OGG1 and other base excision repair factors to chromatin upon oxidative stress. The association of OGG1 with euchromatin is necessary for the removal of 8-oxoG. Mediator subunits CDK8 and MED12 bind to chromatin and interact with OGG1 in response to oxidative stress, suggesting they participate in the recruitment of the DNA glycosylase. The oxidative stress-induced association between the cohesin and mediator complexes and OGG1 reveals an unsuspected function of those complexes in the maintenance of genomic stability.

Prion protein deficiency impairs hematopoietic stem cell determination and sensitizes myeloid progenitors to irradiation.

Highly conserved among species and expressed in various types of cells, numerous roles have been attributed to the cellular prion protein (PrPC). In hematopoiesis, PrPC regulates hematopoietic stem cell self-renewal but the mechanisms involved in this regulation are unknown. Here we show that PrPC regulates hematopoietic stem cell number during aging and their determination towards myeloid progenitors. Furthermore, PrPC protects myeloid progenitors against the cytotoxic effects of total body irradiation. This radioprotective effect was associated with increased cellular prion mRNA level and with stimulation of the DNA repair activity of the Apurinic/pyrimidinic endonuclease 1, a key enzyme of the base excision repair pathway. Altogether, these results show a previously unappreciated role of PrPC in adult hematopoiesis, and indicate that PrPC-mediated stimulation of BER activity might protect hematopoietic progenitors from the cytotoxic effects of total body irradiation.

Identification of the periplasmic DNA receptor for natural transformation of Helicobacter pylori.

Horizontal gene transfer through natural transformation is a major driver of antibiotic resistance spreading in many pathogenic bacterial species. In the case of Gram-negative bacteria, and in particular of Helicobacter pylori, the mechanisms underlying the handling of the incoming DNA within the periplasm are poorly understood. Here we identify the protein ComH as the periplasmic receptor for the transforming DNA during natural transformation in H. pylori. ComH is a DNA-binding protein required for the import of DNA into the periplasm. Its C-terminal domain displays strong affinity for double-stranded DNA and is sufficient for the accumulation of DNA in the periplasm, but not for DNA internalisation into the cytoplasm. The N-terminal region of the protein allows the interaction of ComH with a periplasmic domain of the inner-membrane channel ComEC, which is known to mediate the translocation of DNA into the cytoplasm. Our results indicate that ComH is involved in the import of DNA into the periplasm and its delivery to the inner membrane translocator ComEC.

Correction to: Mutations in the nucleotide binding and hydrolysis domains of helicobacter pylori MutS2 lead to altered biochemical activities and inactivation of its in vivo function.

Following publication of the original article [1], the authors notified us of an error in the presentation of Fig. 6G.

Identification of an XRCC1 DNA binding activity essential for retention at sites of DNA damage.

Repair of two major forms of DNA damage, single strand breaks and base modifications, are dependent on XRCC1. XRCC1 orchestrates these repair processes by temporally and spatially coordinating interactions between several other repair proteins. Here we show that XRCC1 contains a central DNA binding domain (CDB, residues 219-415) encompassing its first BRCT domain. In contrast to the N-terminal domain of XRCC1, which has been reported to mediate damage sensing in vitro, we demonstrate that the DNA binding module identified here lacks binding specificity towards DNA containing nicks or gaps. Alanine substitution of residues within the CDB of XRCC1 disrupt DNA binding in vitro and lead to a significant reduction in XRCC1 retention at DNA damage sites without affecting initial recruitment. Interestingly, reduced retention at sites of DNA damage is associated with an increased rate of repair. These findings suggest that DNA binding activity of XRCC1 plays a significant role in retention at sites of damage and the rate at which damage is repaired.

The C-terminal domain of HpDprA is a DNA-binding winged helix domain that does not bind double-stranded DNA.

DNA-processing protein A, a ubiquitous multidomain DNA-binding protein, plays a crucial role during natural transformation in bacteria. Here, we carried out the structural analysis of DprA from the human pathogen Helicobacter pylori by combining data issued from the 1.8-Å resolution X-ray structure of the Pfam02481 domain dimer (RF), the NMR structure of the carboxy terminal domain (CTD), and the low-resolution structure of the full-length DprA dimer obtained in solution by SAXS. In particular, we sought a molecular function for the CTD, a domain that we show here is essential for transformation in H. pylori. Albeit its structural homology to winged helix DNA-binding motifs, we confirmed that the isolated CTD does not interact with ssDNA nor with dsDNA. The key R52 and K137 residues of RF are crucial for these two interactions. Search for sequences harboring homology to either HpDprA or Rhodopseudomonas palustris DprA CTDs led to the identification of conserved patches in the two CTD. Our structural study revealed the similarity of the structures adopted by these residues in RpDprA CTD and HpDprA CTD. This argues for a conserved, but yet to be defined, CTD function, distinct from DNA binding.

The transcription-coupled DNA repair-initiating protein CSB promotes XRCC1 recruitment to oxidative DNA damage.

Transcription-coupled nucleotide excision repair factor Cockayne syndrome protein B (CSB) was suggested to function in the repair of oxidative DNA damage. However thus far, no clear role for CSB in base excision repair (BER), the dedicated pathway to remove abundant oxidative DNA damage, could be established. Using live cell imaging with a laser-assisted procedure to locally induce 8-oxo-7,8-dihydroguanine (8-oxoG) lesions, we previously showed that CSB is recruited to these lesions in a transcription-dependent but NER-independent fashion. Here we showed that recruitment of the preferred 8-oxoG-glycosylase 1 (OGG1) is independent of CSB or active transcription. In contrast, recruitment of the BER-scaffolding protein, X-ray repair cross-complementing protein 1 (XRCC1), to 8-oxoG lesions is stimulated by CSB and transcription. Remarkably, recruitment of XRCC1 to BER-unrelated single strand breaks (SSBs) does not require CSB or transcription. Together, our results suggest a specific transcription-dependent role for CSB in recruiting XRCC1 to BER-generated SSBs, whereas XRCC1 recruitment to SSBs generated independently of BER relies predominantly on PARP activation. Based on our results, we propose a model in which CSB plays a role in facilitating BER progression at transcribed genes, probably to allow XRCC1 recruitment to BER-intermediates masked by RNA polymerase II complexes stalled at these intermediates.

Mitochondrial maintenance under oxidative stress depends on mitochondrially localised α-OGG1.

Accumulation of 8-oxoguanine (8-oxoG) in mitochondrial DNA and mitochondrial dysfunction have been observed in cells deficient for the DNA glycosylase OGG1 when exposed to oxidative stress. In human cells, up to eight mRNAs for OGG1 can be generated by alternative splicing and it is still unclear which of them codes for the protein that ensures the repair of 8-oxoG in mitochondria. Here, we show that the α-OGG1 isoform, considered up to now to be exclusively nuclear, has a functional mitochondrial-targeting sequence and is imported into mitochondria. We analyse the sub-mitochondrial localisation of α-OGG1 with unprecedented resolution and show that this DNA glycosylase is associated with DNA in mitochondrial nucleoids. We show that the presence of α-OGG1 inside mitochondria and its enzymatic activity are required to preserve the mitochondrial network in cells exposed to oxidative stress. Altogether, these results unveil a new role of α-OGG1 in the mitochondria and indicate that the same isoform ensures the repair of 8-oxoG in both nuclear and mitochondrial genomes. The activity of α-OGG1 in mitochondria is sufficient for the recovery of organelle function after oxidative stress.

Structural basis for the substrate selectivity of Helicobacter pylori NucT nuclease activity.

The Phospholipase D (PLD) superfamily of proteins includes a group of enzymes with nuclease activity on various nucleic acid substrates. Here, with the aim of better understanding the substrate specificity determinants in this subfamily, we have characterised the enzymatic activity and the crystal structure of NucT, a nuclease implicated in Helicobacter pylori purine salvage and natural transformation and compared them to those of its bacterial and mammalian homologues. NucT exhibits an endonuclease activity with a strong preference for single stranded nucleic acids substrates. We identified histidine124 as essential for the catalytic activity of the protein. Comparison of the NucT crystal structure at 1.58 Å resolution reported here with those of other members of the sub-family suggests that the specificity of NucT for single-stranded nucleic acids is provided by the width of a positively charged groove giving access to the catalytic site.

Incorporating interaction networks into the determination of functionally related hit genes in genomic experiments with Markov random fields.

MOTIVATION: Incorporating gene interaction data into the identification of 'hit' genes in genomic experiments is a well-established approach leveraging the 'guilt by association' assumption to obtain a network based hit list of functionally related genes. We aim to develop a method to allow for multivariate gene scores and multiple hit labels in order to extend the analysis of genomic screening data within such an approach. RESULTS: We propose a Markov random field-based method to achieve our aim and show that the particular advantages of our method compared with those currently used lead to new insights in previously analysed data as well as for our own motivating data. Our method additionally achieves the best performance in an independent simulation experiment. The real data applications we consider comprise of a survival analysis and differential expression experiment and a cell-based RNA interference functional screen. AVAILABILITY AND IMPLEMENTATION: We provide all of the data and code related to the results in the paper. CONTACT: sean.j.robinson@utu.fi or laurent.guyon@cea.fr. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

ComB proteins expression levels determine Helicobacter pylori competence capacity.

Helicobacter pylori chronically colonises half of the world's human population and is the main cause of ulcers and gastric cancers. Its prevalence and the increase in antibiotic resistance observed recently reflect the high genetic adaptability of this pathogen. Together with high mutation rates and an efficient DNA recombination system, horizontal gene transfer through natural competence makes of H. pylori one of the most genetically diverse bacteria. We show here that transformation capacity is enhanced in strains defective for recN, extending previous work with other homologous recombination genes. However, inactivation of either mutY or polA has no effect on DNA transformation, suggesting that natural competence can be boosted in H. pylori by the persistence of DNA breaks but not by enhanced mutagenesis. The transformation efficiency of the different DNA repair impaired strains correlates with the number of transforming DNA foci formed on the cell surface and with the expression of comB8 and comB10 competence genes. Overexpression of the comB6-B10 operon is sufficient to increase the transformation capacity of a wild type strain, indicating that the ComB complex, present in the bacterial wall and essential for DNA uptake, can be a limiting factor for transformation efficiency.

A threshold of endogenous stress is required to engage cellular response to protect against mutagenesis.

Endogenous stress represents a major source of genome instability, but is in essence difficult to apprehend. Incorporation of labeled radionuclides into DNA constitutes a tractable model to analyze cellular responses to endogenous attacks. Here we show that incorporation of [(3)H]thymidine into CHO cells generates oxidative-induced mutagenesis, but, with a peak at low doses. Proteomic analysis showed that the cellular response differs between low and high levels of endogenous stress. In particular, these results confirmed the involvement of proteins implicated in redox homeostasis and DNA damage signaling pathways. Induced-mutagenesis was abolished by the anti-oxidant N-acetyl cysteine and plateaued, at high doses, upon exposure to L-buthionine sulfoximine, which represses cellular detoxification. The [(3)H]thymidine-induced mutation spectrum revealed mostly base substitutions, exhibiting a signature specific for low doses (GC > CG and AT > CG). Consistently, the enzymatic activity of the base excision repair protein APE-1 is induced at only medium or high doses. Collectively, the data reveal that a threshold of endogenous stress must be reached to trigger cellular detoxification and DNA repair programs; below this threshold, the consequences of endogenous stress escape cellular surveillance, leading to high levels of mutagenesis. Therefore, low doses of endogenous local stress can jeopardize genome integrity more efficiently than higher doses.

Following transforming DNA in Helicobacter pylori from uptake to expression.

Natural transformation is a potent driver for genetic diversification in bacterial populations. It involves exogenous DNA binding, uptake, transport and internalization into the cytoplasm, where DNA can be processed and integrated into the host chromosome. Direct visualisation of transforming DNA (tDNA) has been limited to its binding to the surface or, in the case of Gram-negative species, to its entrance into the periplasm. We present here for the first time the direct visualisation of tDNA entering the bacterial cytoplasm. We used as a model the Gram-negative pathogen Helicobacter pylori, characterised by a large intraspecies variability that results from high mutation rates and efficient horizontal gene transfer. Using fluorescently labelled DNA, we followed for up to 3 h the fate of tDNA foci formed in the periplasm and eventually internalised into the cytoplasm. By tracking at the single cell level the expression of a fluorescent protein coded by the tDNA, we show that up to 50% of the cells express the transforming phenotype. The overall transformation process in H. pylori, from tDNA uptake to expression of the recombinant gene, can take place in less than 1 h, without requiring a growth arrest, and prior to the replication of the chromosome.