38 research outputs found
Expression of 8-oxoguanine DNA glycosylase (Ogg1) in mouse retina
International audienc
Phi-score: A cell-to-cell phenotypic scoring method for sensitive and selective hit discovery in cell-based assays
International audiencePhenotypic screening monitors phenotypic changes induced by perturbations, including those generated by drugs or RNA interference. Currently-used methods for scoring screen hits have proven to be problematic, particularly when applied to physiologically relevant conditions such as low cell numbers or inefficient transfection. Here, we describe the Phi-score, which is a novel scoring method for the identification of phenotypic modifiers or hits in cell-based screens. Phi-score performance was assessed with simulations, a validation experiment and its application to gene identification in a large-scale RNAi screen. Using robust statistics and a variance model, we demonstrated that the Phi-score showed better sensitivity, selectivity and reproducibility compared to classical approaches. The improved performance of the Phi-score paves the way for cell-based screening of primary cells, which are often difficult to obtain from patients in sufficient numbers. We also describe a dedicated merging procedure to pool scores from small interfering RNAs targeting the same gene so as to provide improved visualization and hit selection
Oxidative stress triggers the preferential assembly of base excision repair complexes on open chromatin regions
How DNA repair machineries detect and access, within the context of chromatin, lesions inducing little or no distortion of the DNA structure is a poorly understood process. Removal of oxidized bases is initiated by a DNA glycosylase that recognises and excises the damaged base, initiating the base excision repair (BER) pathway. We show that upon induction of 8-oxoguanine, a mutagenic product of guanine oxidation, the mammalian 8-oxoguanine DNA glycosylase OGG1 is recruited together with other proteins involved in BER to euchromatin regions rich in RNA and RNA polymerase II and completely excluded from heterochromatin. The underlying mechanism does not require direct interaction of the protein with the oxidized base, however, the release of the protein from the chromatin fraction requires completion of repair. Inducing chromatin compaction by sucrose results in a complete but reversible inhibition of the in vivo repair of 8-oxoguanine. We conclude that after induction of oxidative DNA damage, the DNA glycosylase is actively recruited to regions of open chromatin allowing the access of the BER machinery to the lesions, suggesting preferential repair of active chromosome regions
Human hematopoietic stem/progenitor cells display reactive oxygen species-dependent long-term hematopoietic defects after exposure to low doses of ionizing radiations
Hematopoietic stem cells are responsible for life-long blood cell production and are highly sensitive to exogenous stresses. The effects of low doses of ionizing radiations on radiosensitive tissues such as the hematopoietic tissue are still unknown despite their increasing use in medical imaging. Here, we study the consequences of low doses of ionizing radiations on differentiation and self-renewal capacities of human primary hematopoietic stem/progenitor cells (HSPC). We found that a single 20 mGy dose impairs the hematopoietic reconstitution potential of human HSPC but not their differentiation properties. In contrast to high irradiation doses, low doses of irradiation do not induce DNA double strand breaks in HSPC but, similar to high doses, induce a rapid and transient increase of reactive oxygen species (ROS) that promotes activation of the p38MAPK pathway. HSPC treatment with ROS scavengers or p38MAPK inhibitor prior exposure to 20 mGy irradiation abolishes the 20 mGy-induced defects indicating that ROS and p38MAPK pathways are transducers of low doses of radiation effects. Taken together, these results show that a 20 mGy dose of ionizing radiation reduces the reconstitution potential of HSPC suggesting an effect on the self-renewal potential of human hematopoietic stem cells and pinpointing ROS or the p38MAPK as therapeutic targets. Inhibition of ROS or the p38MAPK pathway protects human primary HSPC from low-dose irradiation toxicity
Distinct spatiotemporal patterns and PARP dependence of XRCC1 recruitment to single-strand break and base excision repair
Single-strand break repair (SSBR) and base excision
repair (BER) of modified bases and abasic sites
share several players. Among them is XRCC1, an
essential scaffold protein with no enzymatic
activity, required for the coordination of both
pathways. XRCC1 is recruited to SSBR by PARP-1,
responsible for the initial recognition of the break.
The recruitment of XRCC1 to BER is still poorly
understood. Here we show by using both local and
global induction of oxidative DNA base damage that
XRCC1 participation in BER complexes can be distinguished
from that in SSBR by several criteria. We
show first that XRCC1 recruitment to BER is independent
of PARP. Second, unlike SSBR complexes
that are assembled within minutes after global
damage induction, XRCC1 is detected later in BER
patches, with kinetics consistent with the repair of
oxidized bases. Third, while XRCC1-containing foci
associated with SSBR are formed both in eu- and
heterochromatin domains, BER complexes are
assembled in patches that are essentially excluded
from heterochromatin and where the oxidized bases
are detected
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
Enod40, a Short Open Reading Frame–Containing mRNA, Induces Cytoplasmic Localization of a Nuclear RNA Binding Protein in Medicago truncatula
In eukaryotes, diverse mRNAs containing only short open reading frames (sORF-mRNAs) are induced at specific stages of development. Their mechanisms of action may involve the RNA itself and/or sORF-encoded oligopeptides. Enod40 genes code for highly structured plant sORF-mRNAs involved in root nodule organogenesis. A novel RNA binding protein interacting with the enod40 RNA, MtRBP1 (for Medicago truncatula RNA Binding Protein 1), was identified using a yeast three-hybrid screening. Immunolocalization studies and use of a MtRBP1-DsRed2 fluorescent protein fusion showed that MtRBP1 localized to nuclear speckles in plant cells but was exported into the cytoplasm during nodule development in enod40-expressing cells. Direct involvement of the enod40 RNA in MtRBP1 relocalization into cytoplasmic granules was shown using a transient expression assay. Using a (green fluorescent protein)/MS2 bacteriophage system to tag the enod40 RNA, we detected in vivo colocalization of the enod40 RNA and MtRBP1 in these granules. This in vivo approach to monitor RNA–protein interactions allowed us to demonstrate that cytoplasmic relocalization of nuclear proteins is an RNA-mediated cellular function of a sORF-mRNA
Lost in the Crowd: How Does Human 8-Oxoguanine DNA Glycosylase 1 (OGG1) Find 8-Oxoguanine in the Genome?
International audienceThe most frequent DNA lesion resulting from an oxidative stress is 7,8-dihydro-8-oxoguanine (8-oxoG). 8-oxoG is a premutagenic base modification due to its capacity to pair with adenine. Thus, the repair of 8-oxoG is critical for the preservation of the genetic information. Nowadays, 8-oxoG is also considered as an oxidative stress-sensor with a putative role in transcription regulation. In mammalian cells, the modified base is excised by the 8-oxoguanine DNA glycosylase (OGG1), initiating the base excision repair (BER) pathway. OGG1 confronts the massive challenge that is finding rare occurrences of 8-oxoG among a million-fold excess of normal guanines. Here, we review the current knowledge on the search and discrimination mechanisms employed by OGG1 to find its substrate in the genome. While there is considerable data from in vitro experiments, much less is known on how OGG1 is recruited to chromatin and scans the genome within the cellular nucleus. Based on what is known of the strategies used by proteins searching for rare genomic targets, we discuss the possible scenarios allowing the efficient detection of 8-oxoG by OGG1