Conditional degradation of SDE2 by the Arg/N-End rule pathway regulates stress response at replication forks

Multiple pathways counteract DNA replication stress to prevent genomic instability and tumorigenesis. The recently identified human SDE2 is a genome surveillance protein regulated by PCNA, a DNA clamp and processivity factor at replication forks. Here, we show that SDE2 cleavage after its ubiquitin-like domain generates Lys-SDE2^(Ct), the C-terminal SDE2 fragment bearing an N-terminal Lys residue. Lys-SDE2^(Ct) constitutes a short-lived physiological substrate of the Arg/N-end rule proteolytic pathway, in which UBR1 and UBR2 ubiquitin ligases mediate the degradation. The Arg/N-end rule and VCP/p97^(UFD1-NPL4) segregase cooperate to promote phosphorylation-dependent, chromatin-associated Lys-SDE2^(Ct) degradation upon UVC damage. Conversely, cells expressing the degradation-refractory K78V mutant, Val-SDE2^(Ct), fail to induce RPA phosphorylation and single-stranded DNA formation, leading to defects in PCNA-dependent DNA damage bypass and stalled fork recovery. Together, our study elucidates a previously unappreciated axis connecting the Arg/N-end rule and the p97-mediated proteolysis with the replication stress response, working together to preserve replication fork integrity

Conditional degradation of SDE2 by the Arg/N-End rule pathway regulates stress response at replication forks

Multiple pathways counteract DNA replication stress to prevent genomic instability and tumorigenesis. The recently identified human SDE2 is a genome surveillance protein regulated by PCNA, a DNA clamp and processivity factor at replication forks. Here, we show that SDE2 cleavage after its ubiquitin-like domain generates Lys-SDE2^(Ct), the C-terminal SDE2 fragment bearing an N-terminal Lys residue. Lys-SDE2^(Ct) constitutes a short-lived physiological substrate of the Arg/N-end rule proteolytic pathway, in which UBR1 and UBR2 ubiquitin ligases mediate the degradation. The Arg/N-end rule and VCP/p97^(UFD1-NPL4) segregase cooperate to promote phosphorylation-dependent, chromatin-associated Lys-SDE2^(Ct) degradation upon UVC damage. Conversely, cells expressing the degradation-refractory K78V mutant, Val-SDE2^(Ct), fail to induce RPA phosphorylation and single-stranded DNA formation, leading to defects in PCNA-dependent DNA damage bypass and stalled fork recovery. Together, our study elucidates a previously unappreciated axis connecting the Arg/N-end rule and the p97-mediated proteolysis with the replication stress response, working together to preserve replication fork integrity

SDE2 integrates into the TIMELESS-TIPIN complex to protect stalled replication forks

Protecting replication fork integrity during DNA replication is essential for maintaining genome stability. Here, we report that SDE2, a PCNA-associated protein, plays a key role in maintaining active replication and counteracting replication stress by regulating the replication fork protection complex (FPC). SDE2 directly interacts with the FPC component TIMELESS (TIM) and enhances its stability, thereby aiding TIM localization to replication forks and the coordination of replisome progression. Like TIM deficiency, knockdown of SDE2 leads to impaired fork progression and stalled fork recovery, along with a failure to activate CHK1 phosphorylation. Moreover, loss of SDE2 or TIM results in an excessive MRE11-dependent degradation of reversed forks. Together, our study uncovers an essential role for SDE2 in maintaining genomic integrity by stabilizing the FPC and describes a new role for TIM in protecting stalled replication forks. We propose that TIM-mediated fork protection may represent a way to cooperate with BRCA-dependent fork stabilization. The fork protection complex (FPC), including the proteins TIMELESS and TIPIN, stabilizes the replisome to ensure unperturbed fork progression during DNA replication. Here the authors reveal that that SDE2, a PCNA-associated protein, plays an important role in maintaining active replication and protecting stalled forks by regulating the replication fork protection complex (FPC)

Étude des propriétés superficielles de quelques oxydes métalliques par chromatographie gazeuse inverse et par zétamétrie en milieux aqueux et organique

Inverse gas chromatography and zeta potential techniques are usedito study some of the surface properties of zinc oxide ZnO, monogai (essentially constituted; by ZnO), zinc hydroxide Zn (OH)2 and magnesium oxide MgO. The dispersive component of their surface energy,; their (Lewis) acide-base characteristics and isoelectic point (IEP) in both aqueous and organic-media, are determined in order to understand and predict the adhesive capacity of such oxides. We showed that the dispersive component of the surface energy γSD of these oxides depends; on temperature T ; irregularly for monogai and linearly for ZnO and Zn (OH)2- For MgO) γSD remains constant as a function of the temperature. It is shown, that Zn (OH)O exhibits the highest surface acidity followed respectively in decreasing acidity order by monogai, ZnO and MgO. Zeta potential measurements of the oxides in aqueous medium lead to the conclusion that MgO is more basic (IEP = 11) than the other oxides, whereas monogai is the most acidic one, in tema of Brönsted acide-base concept

Étude des propriétés superficielles de quelques oxydes métalliques par chromatographie gazeuse inverse et par zétamétrie en milieux aqueux et organique

Les résultats obtenus par chromatographie gazeuse inverse montrent que les composantes dispersives de l’énergie de surface du monogai (contenant de l’oxyde de zinc en surface) et de l’oxyde de zinc, sont toutes deux voisines de 80 mJ/m2 à la température ambiante et diminuent linéairement avec la température. La composante dispersive de l’énergie de surface (γSD) de Zn(OH)2 est plus élevée (170 mJ/m2) et décroît aussi linéairement avec la température. Cependant, le γSD de l’oxyde de magnésium ne varie pas avec la température et se maintient autour d’une valeur moyenne de 45 mJ/m2. Nous avons observé que le monogai, ZnO et Zn(OH)2 interagissent avec les bases, les acides et les sondes amphotères. Ils ont donc une surface amphotère.En milieu organique, les mesures du potentiel zêta montrent que les deux oxydes ZnO et MgO ont le même comportement envers les divers solvants. Cependant, MgO est chargé plus positivement dans les solvants acides et moins négativement dans les solvants basiques. Il donne plus d’électrons aux solvants acides et en reçoit moins des solvants basiques. De même dans les solvants amphotères, ZnO reçoit plus de charges négatives que MgO. Ce dernier est donc légèrement plus basique que ZnO au sens de Lewis.En étudiant le potentiel zéta des différents oxydes en milieu aqueux en fonction du pH, nous avons observé que le monogai possède un point isoélectrique (PIE) de pH = 4,7 alorss que celui de Zn(QH)22. est de 6. Ces deux matériaux présentent donc une surface acide. La surface de l’oxyde de zinc est neutre (PIE = 7,2). Enfin, l’oxyde de magnésium se distingue nettement des autres produits par la très forte basicité de sa surface (PIE = 11)

Étude des interactions spécifiques entre certains oxydes métalliques et des molécules organiques modèles

Dans une étude précédente, nous avons étudié le comportement acido-basique des oxydes ou hydroxydes métalliques tels que MgO, ZnO, Zn(OH)2, Al2O3 et le monogai, par des mesures de potentiel zêta en milieu organique et en milieu aqueux.Nous nous intéressons, dans cette étude, à la caractérisation acido-basique de ces oxydes par chromatographie gazeuse inverse à dilution infinie en déterminant les interactions spécifiques échangées entre les solides utilisés et des molécules organiques modèles. Le caractère amphotère des différents oxydes a été prouvé en calculant leurs constantes d'acidité et de basicité. L'oxyde de magnésium est le plus basique alors que l'oxyde de zinc a plutôt un caractère acide.Nous avons également établi une nouvelle méthode permettant le calcul des constantes d'acidité Ka et de basicité Kd d'un solide en proposant une correction à la relation classique qui relie la variation d'enthalpie spécifique (-AHSP) d'adsorption des molécules polaires sur les différents solides aux nombres donneur ND et accepteur NA de la sonde gazeuse. Un nouveau paramètre K traduisant le caractère amphotère d'un solide a été proposé :(-ΔHSP) = KA.ND + KD.NA – K. NA.NDNous avons constaté que notre modèle donne des résultats meilleurs que ceux obtenus avec la formule classique

Étude des interactions spécifiques entre certains oxydes métalliques et des molécules organiques modèles

In a previous paper, we studied the acid-base behaviour of some metallic oxides (ZnO, Zn (OH)2, MgO, etc.) in organic or aqueous media by measuring their zeta potential. In this study , we are interested in the acid-base characterization of these oxides by using inverse gas chromatography technique. We determined the specific interactions between them and model organic molecules and proved the amphoteric feature of such oxides by calculating their acidic and basic parameters. It was proved that MgO is more basic than the other oxides and ZnO is rather acidic. We propose a new method to calculate acidic Ka and basic Kd parameters of a solid by adding a third parameter K in the relationship that related specific enthalpy variation (-AHSP) of polar molecules to their acceptor number AN and their donor number DN. The parameter K can reflect the amphoteric feature of a solid. The proposed relationship is : (-ΔHSP) – KA-ND + KD.NA – K. NA.ND We prove that our model gives the best results relatively to those obtained by the classic equation

Poly(ADP-ribosyl)ation of TIMELESS limits DNA replication stress and promotes stalled fork protection

Summary: Poly(ADP-ribosyl)ation (PARylation), catalyzed mainly by poly(ADP-ribose) polymerase (PARP)1, is a key posttranslational modification involved in DNA replication and repair. Here, we report that TIMELESS (TIM), an essential scaffold of the replisome, is PARylated, which is linked to its proteolysis. TIM PARylation requires recognition of auto-modified PARP1 via two poly(ADP-ribose)-binding motifs, which primes TIM for proteasome-dependent degradation. Cells expressing the PARylation-refractory TIM mutant or under PARP inhibition accumulate TIM at DNA replication forks, causing replication stress and hyper-resection of stalled forks. Mechanistically, aberrant engagement of TIM with the replicative helicase impedes RAD51 loading and protection of reversed forks. Accordingly, defective TIM degradation hypersensitizes BRCA2-deficient cells to replication damage. Our study defines TIM as a substrate of PARP1 and elucidates how the control of replisome remodeling by PARylation is linked to stalled fork protection. Therefore, we propose a mechanism of PARP inhibition that impinges on the DNA replication fork instability caused by defective TIM turnover

DNA Adduct Formation of 4-Aminobiphenyl and Heterocyclic Aromatic Amines in Human Hepatocytes.

DNA adduct formation of the aromatic amine, 4-aminobiphenyl (4-ABP), a known human carcinogen present in tobacco smoke, and the heterocyclic aromatic amines (HAAs), 2-amino-9H-pyrido[2,3-b]indole (AαC), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), potential human carcinogens, which are also present in tobacco smoke or formed during the high-temperature cooking of meats, was investigated in freshly cultured human hepatocytes. The carcinogens (10 μM) were incubated with hepatocytes derived from eight different donors for time periods up to 24 h. The DNA adducts were quantified by liquid chromatography-electrospray ionization mass spectrometry with a linear quadrupole ion trap mass spectrometer. The principal DNA adducts formed for all of the carcinogens were N-(deoxyguanosin-8-yl) (dG-C8) adducts. The levels of adducts ranged from 3.4 to 140 adducts per 10(7) DNA bases. The highest level of adduct formation occurred with AαC, followed by 4-ABP, then by PhIP, MeIQx, and IQ. Human hepatocytes formed dG-C8-HAA-adducts at levels that were up to 100-fold greater than the amounts of adducts produced in rat hepatocytes. In contrast to HAA adducts, the levels of dG-C8-4-ABP adduct formation were similar in human and rat hepatocytes. These DNA binding data demonstrate that the rat, an animal model that is used for carcinogenesis bioassays, significantly underestimates the potential hepatic genotoxicity of HAAs in humans. The high level of DNA adducts formed by AαC, a carcinogen produced in tobacco smoke at levels that are up to 100-fold higher than the amounts of 4-ABP, is noteworthy. The possible causal role of AαC in tobacco-associated cancers warrants investigation