PARP-1 activation regulates the DNA damage response to DNA double-strand breaks

Les cassures double-brin de l'ADN, lorsque incorrectement réparées, peuvent avoir des conséquences fatales telles que des délétions et des réarrangements chromosomiques, favorisant la carcinogenèse. La poly(ADP-ribosyl)ation réalisée par la protéine poly(ADP-ribose) polymérase-1 (PARP-1) est l'une des premières modifications post-traductionnelles qui se produisent en réponse aux dommages à l'ADN. La PARP-1 utilise la nicotinamide pour générer un polymère chargé négativement, nommé poly(ADP-ribose) polymère (PAR), lequel est attaché en majorité à la PARP-1 elle-même ainsi qu'à d'autres protéines cibles. Le PAR a récemment été reconnu comme un signal de recrutement pour certaines protéines de réparation aux sites de dommages à l'ADN, mais un débat est en cours quant au rôle précis de la PARP-1 et du PAR dans la réponse aux dommages de l'ADN. Au cours de mon projet de doctorat, nous avons pu confirmer que les protéines qui se retrouvent en complexe avec le PAR immédiatement après les dommages à l'ADN sont principalement des facteurs de réparation. Étonnamment, les complexes protéiques associés au PAR pendant la période de récupération suite aux dommages sont enrichis en facteurs de liaison à l'ARN. Toutefois, la protéine liant l'ARN la plus abondante que nous avons détectée dans l'interactome du PAR, soit NONO, ne suit pas cette dernière cinétique puisqu'elle est fortement enrichie immédiatement après les dommages à l'ADN. Notre étude subséquente de NONO dans la réponse aux cassures double-brin de l'ADN a étonnamment révélé une implication directe de celle-ci par le mécanismede réparation de jonction des extrémités non-homologues. En plus, nous avons constaté que NONO se lie fortement et spécifiquement au PAR via son motif 1 de la reconnaissance de l'ARN, soulignant la compétition entre les PAR et l'ARN pour le même site de liaison. Fait intéressant, le recrutement in vivo de NONO aux sites de dommages de l'ADN dépend entièrement du PAR et nécessite le motif 1 de la reconnaissance de l'ARN. En conclusion, nos résultats établissent NONO comme une nouvelle protéine impliquée dans la réponse aux cassures double-brin de l'ADN et plus généralement démontrent un autre niveau de complexité supplémentaire dans l'interdépendance de la biologie de l'ARN et la réparation de l'ADN.DNA double-strand breaks are potentially lethal lesions, which if not repaired correctly, can have harmful consequences such as carcinogenesis promoted by chromosome deletions and rearrangements. Poly(ADP-ribosyl)ation carried out by poly(ADP-ribose) polymerase 1 (PARP-1) is one of the first posttranslational modifications occurring in response to DNA damage. In brief, PARP-1 uses nicotinamide to generate a negatively charged polymer called poly(ADP-ribose) polymer (PAR), that can be attached to acceptor proteins, which is to a large extent PARP-1 itself. PAR has recently been recognized as a recruitment signal for key DNA repair proteins to sites of DNA damage but the precise role of PARP-1 and its catalytic product PAR in the DNA damage response are still a matter of ongoing debate. Throughout my doctoral work, we confirmed that the proteins in complex with PAR promptly after DNA damage are mostly DNA repair proteins, whereas during the period of recovery from DNA damage, the PAR interactome is highly enriched with RNA processing factors. Interestingly, one of the most abundant RNA-binding proteins detected in the PAR interactome, namely NONO, did not follow these kinetics as it was highly enriched immediately after DNA damage in the DNA repair protein complexes centered on PAR. Our subsequent investigation of NONO in the DNA damage response to double-strand breaks strikingly revealed a direct implication for NONO in repair by nonhomologous end joining (NHEJ). Moreover, we found that NONO strongly and specifically binds to PAR through its RNA-recognition motif 1 (RRM1), highlighting competition between PAR and RNA for the same binding site. Remarkably, the in vivo recruitment of NONO to DNA damage sites completely depends on PAR and requires the RRM1 motif. In conclusion, our results establish NONO as a new protein implicated in the DNA damage response to double-strand break and in broader terms add another layer of complexity to the cross-talk between RNA-biology and DNA repair

Mating behaviour of two polygamous shorebird species in the Arctic

The mating behaviour is a critical part of an individual’s life, given that it directly influences the reproductive success of sexually reproducing organisms. In nature, we can observe a fascinating diversity of mating strategies shaped by sexual and natural selection. Understanding the evolution of this diversity is a central part of evolutionary biology. Nevertheless, the underlying evolutionary and ecological factors that shape the evolution of this diversity are still not fully understood, and on top of that we even lack detailed knowledge of the mating behaviour for many species. In this dissertation, we enhance our understanding of the mating behaviours and strategies of two polygamous shorebird species. The focus of our studies are the polyandrous red phalarope Phalaropus fulicarius and the polygynous pectoral sandpiper Calidris melanotos, two sympatric breeders of the Arctic tundra with distinctly different mating systems. More specifically, we investigated the social and genetic mating system of the red phalarope, by quantifying the number of social and genetic mates and describing the copulation behaviour in this context. This allowed us to test the “sperm storage hypothesis”, which predicts that extra-pair paternity in sequentially polyandrous species is mainly the result of sperm stored by females from within-pair copulations with a previous mate. Next, we investigated mate guarding behavior in red phalaropes under consideration of the male and female perspective and in relation to breeding phenology, time relative to mean clutch initiation, and to other mutually exclusive behaviours, like incubation or mate searching. Furthermore, we investigated if the apparently nomadic movements of pectoral sandpipers between potential breeding sites are influenced by the prevailing wind conditions. Finally, I describe the mating system of both species in detail, discuss how sexual selection shapes the mating strategies in both species and sexes, and discuss how environmental conditions influence mating strategies. To accomplish this, we recorded in great detail the mating behaviour of red phalaropes during the time span of three breeding seasons in Utqiaġvik, Alaska. We caught and colour-banded nearly all individuals and documented all breeding attempts within our study site. Eggs were collected and artificially incubated to prevent data loss due to predation events, which allowed us to perform comprehensive paternity analysis. Furthermore, modern tracking devices allowed us to follow individual movements and pair-wise association patterns continuously, which allowed us to study variation in mate guarding behaviour. Additionally, we used a previously published data set of pectoral sandpiper movements from two breeding season, in combination with wind data from a global reanalysis model to investigate the influence of wind conditions on breeding site sampling behaviour. We found lower rates of social polyandry and genetic polyandry in red phalaropes than previous studies reported. Overall, 7% of females (11/162) had multiple social mates and extra-pair paternity occurred in 11% (37/334) of the nests. Our paternity analysis and behavioural observations provide limited evidence for the sperm storage hypothesis. Our findings indicate that stored sperm from a previous mate does not significantly contribute to extra-pair paternity in this sequentially polyandrous species. Instead, extra-pair paternity was generally due to two mechanisms: firstly to extra-pair copulations by both sexes during the period between pair establishment and early incubation; then to rapid mate switching by females in the context of attempts to acquire multiple care-giving males. We show that red phalarope pairs were almost continuously together in the days before clutch initiation and showed no sex-bias in separation movements, which suggests that both pair members guard their mate. Still, limited sexual conflict arises through biases in the operational sex-ratio and a trade-off with male nest attendance. We found no clear relationship between mate guarding intensity and the occurrence of extra-pair paternity. Our analysis on the breeding site sampling behaviour of pectoral sandpipers suggests that the wind conditions influence movements in two ways. First, stronger wind support led to increased ground speed and was associated with a longer flight range, and second, males had a higher chance of flying in the direction with more favourable wind conditions. In conclusion, we found that extra-pair paternity in red phalaropes can mainly be explained by female strategies to acquire multiple mates and that in this non-territorial socially polyandrous species, mutual benefits of mate guarding might be the process underlying the evolution of a brief but strong social pair bond, with the unique purpose of producing a clutch for a care-giving male. Polygamy in both species is likely influenced by the length of the breeding season and its spatiotemporal variation throughout the breeding range, as well as local operational sex ratios. Large scale breeding site sampling behaviour can be influenced by the prevailing wind conditions in pectoral sandpipers and consequently effect local breeding densities. Both species are characterised by strong intrasexual selection and direct fitness benefits seem to play a more important role than indirect fitness benefits in mate choice. Still, it is necessary to consider the interplay of intrasexual selection and mate choice to completely understand the factors shaping sexual selection, especially in the context of sex-specific biases in the operational sex ratio and individual strategies to maximise reproductive success from the male and female perspective

Sexual dimorphism in glioma glycolysis underlies sex differences in survival

The molecular bases for sex differences in cancer remain undefined and how to incorporate them into risk stratification remains undetermined. Given sex differences in metabolism and the inverse correlation between fluorodeoxyglucose (FDG) uptake and survival, we hypothesized that glycolytic phenotyping would improve glioma subtyping. Using retrospectively acquired lower-grade glioma (LGG) transcriptome data from The Cancer Genome Atlas (TCGA), we discovered male-specific decreased survival resulting from glycolytic gene overexpression. Patients within this high-glycolytic group showed significant differences in the presence of key genomic alterations (i.e., 1p/19q codeletion, CIC, EGFR, NF1, PTEN, FUBP1, and IDH mutations) compared with the low-glycolytic group. Although glycolytic stratification defined poor prognostic males independent of grade, histology, TP53, and ATRX mutation status, we unexpectedly found that females with high-glycolytic gene expression and wild-type IDH survived longer than all other wild-type patients. Validation with an independent metabolomics dataset from grade 2 gliomas determined that glycolytic metabolites selectively stratified males and also uncovered a potential sexual dimorphism in pyruvate metabolism. These findings identify a potential synergy between patient sex, tumor metabolism, and genomic alterations in determining outcome for glioma patients

Consistent variation in individual migration strategies of brown skuas

Seabirds show remarkable variability in migration strategies among individuals and populations. In this study, we analysed 47 migrations of 28 brown skuas Catharacta antarctica lonnbergi breeding on King George Island in the Maritime Antarctic. Brown skuas from this population used a large area during the non-breeding period north of 55°S, including parts of the Patagonian Shelf, Argentine Basin and South Brazil Shelf, areas which are characterised by high levels of marine productivity. However, individual birds utilised only a subset of these areas, adopting 1 of 4 distinct migration strategies to which they were highly faithful between years, and showed high repeatability in departure and arrival dates at the breeding ground. Although they spent the majority of the non-breeding season within a particular region, almost all individuals used the same area in the late winter, exploiting its seasonal peak in productivity. Overall, these results indicate consistent individual variation in migration strategies that may reflect a combination of genetic control and individual experience, but with considerable flexibility to shift distribution in response to prevailing environmental conditions

Experimental Parameter Study and Inherent Scattering of Safety Characteristics of Dusts

Safety characteristics are widely used in industrial processes to avoid explosive atmospheres (primary explosion protection) or to mitigate the consequences of an explosion (constructive explosion protection). Several laboratory parameters influence the determined values when performing the test series such as the beginning pressure and the pre-ignition pressure rise, the ignition source, the ignition energy, the burning duration and volume or the concentration of the combustible substance. In the different standards for the determination of safety characteristics of dusts there is no statement about the scattering or the deviation when parameters are chosen or occur on the borders of their allowed range. Thus, two laboratories might determine values that are hardly comparable for the same given substance. This article summarizes some of the influential factors that cause a deviation and shows the inherent scattering of dust tests when all other parameters are kept constant. It also provides some advice how to minimize the deviation and the scattering with little effort

Histone Ubiquitination by the DNA Damage Response Is Required for Efficient DNA Replication in Unperturbed S Phase

Chromatin ubiquitination by the ubiquitin ligase RNF168 is critical to regulate the DNA damage response (DDR). DDR deficiencies lead to cancer-prone syndromes, but whether this reflects DNA repair defects is still elusive. We identified key factors of the RNF168 pathway as essential mediators of efficient DNA replication in unperturbed S phase. We found that loss of RNF168 leads to reduced replication fork progression and to reversed fork accumulation, particularly evident at repetitive sequences stalling replication. Slow fork progression depends on MRE11-dependent degradation of reversed forks, implicating RNF168 in reversed fork protection and restart. Consistent with regular nucleosomal organization of reversed forks, the replication function of RNF168 requires H2A ubiquitination. As this novel function is shared with the key DDR players ATM, γH2A.X, RNF8, and 53BP1, we propose that double-stranded ends at reversed forks engage classical DDR factors, suggesting an alternative function of this pathway in preventing genome instability and human disease

PARP-1 dependent recruitment of the amyotrophic lateral sclerosis-associated protein FUS/TLS to sites of oxidative DNA damage

Amyotrophic lateral sclerosis (ALS) is associated with progressive degeneration of motor neurons. Several of the genes associated with this disease encode proteins involved in RNA processing, including fused-in-sarcoma/translocated-in-sarcoma (FUS/TLS). FUS is a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family of proteins that bind thousands of pre-mRNAs and can regulate their splicing. Here, we have examined the possibility that FUS is also a component of the cellular response to DNA damage. We show that both GFP-tagged and endogenous FUS re-localize to sites of oxidative DNA damage induced by UVA laser, and that FUS recruitment is greatly reduced or ablated by an inhibitor of poly (ADP-ribose) polymerase activity. Consistent with this, we show that recombinant FUS binds directly to poly (ADP-ribose) in vitro, and that both GFP-tagged and endogenous FUS fail to accumulate at sites of UVA laser induced damage in cells lacking poly (ADP-ribose) polymerase-1. Finally, we show that GFP-FUS(R521G), harbouring a mutation that is associated with ALS, exhibits reduced ability to accumulate at sites of UVA laser-induced DNA damage. Together, these data suggest that FUS is a component of the cellular response to DNA damage, and that defects in this response may contribute to ALS

Nuclear actin polymerization rapidly mediates replication fork remodeling upon stress by limiting PrimPol activity

Cells rapidly respond to replication stress actively slowing fork progression and inducing fork reversal. How replication fork plasticity is achieved in the context of nuclear organization is currently unknown. Using nuclear actin probes in living and fixed cells, we visualized nuclear actin filaments in unperturbed S phase and observed their rapid extension in number and length upon genotoxic treatments, frequently taking contact with replication factories. Chemically or genetically impairing nuclear actin polymerization shortly before these treatments prevents active fork slowing and abolishes fork reversal. Defective fork remodeling is linked to deregulated chromatin loading of PrimPol, which promotes unrestrained and discontinuous DNA synthesis and limits the recruitment of RAD51 and SMARCAL1 to nascent DNA. Moreover, defective nuclear actin polymerization upon mild replication interference induces chromosomal instability in a PRIMPOL-dependent manner. Hence, by limiting PrimPol activity, nuclear F-actin orchestrates replication fork plasticity and is a key molecular determinant in the rapid cellular response to genotoxic treatments