RFWD3-Dependent Ubiquitination of RPA Regulates Repair at Stalled Replication Forks.

PubMed

Elia, Andrew E H; Wang, David C; Willis, Nicholas A; Boardman, Alexander P; Hajdu, Ildiko; Adeyemi, Richard O; Lowry, Elizabeth; Gygi, Steven P; Scully, Ralph; Elledge, Stephen J

2015-10-15

We have used quantitative proteomics to profile ubiquitination in the DNA damage response (DDR). We demonstrate that RPA, which functions as a protein scaffold in the replication stress response, is multiply ubiquitinated upon replication fork stalling. Ubiquitination of RPA occurs on chromatin, involves sites outside its DNA binding channel, does not cause proteasomal degradation, and increases under conditions of fork collapse, suggesting a role in repair at stalled forks. We demonstrate that the E3 ligase RFWD3 mediates RPA ubiquitination. RFWD3 is necessary for replication fork restart, normal repair kinetics during replication stress, and homologous recombination (HR) at stalled replication forks. Mutational analysis suggests that multisite ubiquitination of the entire RPA complex is responsible for repair at stalled forks. Multisite protein group sumoylation is known to promote HR in yeast. Our findings reveal a similar requirement for multisite protein group ubiquitination during HR at stalled forks in mammalian cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Roles of PCNA ubiquitination and TLS polymerases κ and η in the bypass of methyl methanesulfonate-induced DNA damage.

PubMed

Wit, Niek; Buoninfante, Olimpia Alessandra; van den Berk, Paul C M; Jansen, Jacob G; Hogenbirk, Marc A; de Wind, Niels; Jacobs, Heinz

2015-01-01

Translesion synthesis (TLS) provides a highly conserved mechanism that enables DNA synthesis on a damaged template. TLS is performed by specialized DNA polymerases of which polymerase (Pol) κ is important for the cellular response to DNA damage induced by benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), ultraviolet (UV) light and the alkylating agent methyl methanesulfonate (MMS). As TLS polymerases are intrinsically error-prone, tight regulation of their activity is required. One level of control is provided by ubiquitination of the homotrimeric DNA clamp PCNA at lysine residue 164 (PCNA-Ub). We here show that Polκ can function independently of PCNA modification and that Polη can function as a backup during TLS of MMS-induced lesions. Compared to cell lines deficient for PCNA modification (Pcna(K164R)) or Polκ, double mutant cell lines display hypersensitivity to MMS but not to BPDE or UV-C. Double mutant cells also displayed delayed post-replicative TLS, accumulate higher levels of replication stress and delayed S-phase progression. Furthermore, we show that Polη and Polκ are redundant in the DNA damage bypass of MMS-induced DNA damage. Taken together, we provide evidence for PCNA-Ub-independent activation of Polκ and establish Polη as an important backup polymerase in the absence of Polκ in response to MMS-induced DNA damage. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

The dynamics of histone H2A ubiquitination in HeLa cells exposed to rapamycin, ethanol, hydroxyurea, ER stress, heat shock and DNA damage.

PubMed

Nakata, Shiori; Watanabe, Tadashi; Nakagawa, Koji; Takeda, Hiroshi; Ito, Akihiro; Fujimuro, Masahiro

2016-03-25

Polyubiquitination plays key roles in proteasome-dependent and independent cellular events, whereas monoubiquitination is involved in gene expression, DNA repair, protein-protein interaction, and protein trafficking. We previously developed an FK2 antibody, which specifically recognizes poly-Ub moieties but not free Ub. To elucidate the role of Ub conjugation in response to cellular stress, we used FK2 to investigate whether chemical stress (rapamycin, ethanol, or hydroxyurea), ER stress (thapsigargin or tunicamycin), heat shock or DNA damage (H2O2 or methyl methanesulfonate) affect the formation of Ub conjugates including histone H2A (hH2A) ubiquitination. First, we found that all forms of stress tested increased poly-ubiquitinated proteins in HeLa cells. Furthermore, rapamycin and hydroxyurea treatment, and ER stress increased ubiquitination of hH2A, while methyl methanesulfonate (MMS) treatment induced deubiquitination of hH2A. The ethanol and H2O2 treatments, and heat shock transiently induced hH2A de-ubiquitination, although deubiquitinated hH2A were ubiquitinated again by subsequent cultivation. We also revealed that FK2 reacts with not only polyubiquitinated proteins but also mono-ubiquitinated hH2A. With the exception of MMS, all forms of stress tested increased the acetylation of K5-hH2A, K9-hH3 and K8-hH4 in addition to ubiquitination. K118 and K119 of hH2A were ubiquitinated in cells under normal conditions, and K119 was the major ubiquitination site. The MMS-treatment and heat shock induced the deubiquitination of both K118 and K119-histone H2A. Interestingly, MMS treatment did not affect cell HeLa cell viability expressing double-mutant hH2A (KK118,119AA-hH2A), while heat shock slightly but significantly decreased viability of double-mutant hH2A expressing cells, indicating that ubiquitination of both sites associates with recovery from heat shock but not MMS treatment. Thus, we characterized FK2 reactivity and demonstrated that various stresses alter

A novel regulation mechanism of DNA repair by damage-induced and RAD23-dependent stabilization of xeroderma pigmentosum group C protein

PubMed Central

Ng, Jessica M.Y.; Vermeulen, Wim; van der Horst, Gijsbertus T.J.; Bergink, Steven; Sugasawa, Kaoru; Vrieling, Harry; Hoeijmakers, Jan H.J.

2003-01-01

Primary DNA damage sensing in mammalian global genome nucleotide excision repair (GG-NER) is performed by the xeroderma pigmentosum group C (XPC)/HR23B protein complex. HR23B and HR23A are human homologs of the yeast ubiquitin-domain repair factor RAD23, the function of which is unknown. Knockout mice revealed that mHR23A and mHR23B have a fully redundant role in NER, and a partially redundant function in embryonic development. Inactivation of both genes causes embryonic lethality, but appeared still compatible with cellular viability. Analysis of mHR23A/B double-mutant cells showed that HR23 proteins function in NER by governing XPC stability via partial protection against proteasomal degradation. Interestingly, NER-type DNA damage further stabilizes XPC and thereby enhances repair. These findings resolve the primary function of RAD23 in repair and reveal a novel DNA-damage-dependent regulation mechanism of DNA repair in eukaryotes, which may be part of a more global damage-response circuitry. PMID:12815074

Novel TDP2-ubiquitin interactions and their importance for the repair of topoisomerase II-mediated DNA damage

PubMed Central

Rao, Timsi; Gao, Rui; Takada, Saeko; Al Abo, Muthana; Chen, Xiang; Walters, Kylie J.; Pommier, Yves; Aihara, Hideki

2016-01-01

Tyrosyl DNA phosphodiesterase 2 (TDP2) is a multifunctional protein implicated in DNA repair, signal transduction and transcriptional regulation. In its DNA repair role, TDP2 safeguards genome integrity by hydrolyzing 5′-tyrosyl DNA adducts formed by abortive topoisomerase II (Top2) cleavage complexes to allow error-free repair of DNA double-strand breaks, thereby conferring cellular resistance against Top2 poisons. TDP2 consists of a C-terminal catalytic domain responsible for its phosphodiesterase activity, and a functionally uncharacterized N-terminal region. Here, we demonstrate that this N-terminal region contains a ubiquitin (Ub)-associated (UBA) domain capable of binding multiple forms of Ub with distinct modes of interactions and preference for either K48- or K63-linked polyUbs over monoUb. The structure of TDP2 UBA bound to monoUb shows a canonical mode of UBA-Ub interaction. However, the absence of the highly conserved MGF motif and the presence of a fourth α-helix make TDP2 UBA distinct from other known UBAs. Mutations in the TDP2 UBA-Ub binding interface do not affect nuclear import of TDP2, but severely compromise its ability to repair Top2-mediated DNA damage, thus establishing the importance of the TDP2 UBA–Ub interaction in DNA repair. The differential binding to multiple Ub forms could be important for responding to DNA damage signals under different contexts or to support the multi-functionality of TDP2. PMID:27543075

A Ubiquitin-Proteasome Pathway for the Repair of Topoisomerase I-DNA Covalent Complexes*S⃞

PubMed Central

Lin, Chao-Po; Ban, Yi; Lyu, Yi Lisa; Desai, Shyamal D.; Liu, Leroy F.

2008-01-01

Reversible topoisomerase I (Top1)-DNA cleavage complexes are the key DNA lesion induced by anticancer camptothecins (e.g. topotecan and irinotecan) as well as structurally perturbed DNAs (e.g. oxidatively damaged DNA, UV-irradiated DNA, alkylated DNA, uracil-substituted DNA, mismatched DNA, gapped and nicked DNA, and DNA with abasic sites). Top1 cleavage complexes arrest transcription and trigger transcription-dependent degradation of Top1, a phenomenon termed Top1 down-regulation. In the current study, we have investigated the role of Top1 down-regulation in the repair of Top1 cleavage complexes. Using quiescent (serum-starved) human WI-38 cells, camptothecin (CPT) was shown to induce Top1 down-regulation, which paralleled the induction of DNA single-strand breaks (SSBs) (assayed by comet assays) and ATM autophosphorylation (at Ser-1981). Interestingly, Top1 down-regulation, induction of DNA SSBs and ATM autophosphorylation were all abolished by the proteasome inhibitor MG132. Furthermore, studies using immunoprecipitation and dominant-negative ubiquitin mutants have suggested a specific requirement for the assembly of Lys-48-linked polyubiquitin chains for CPT-induced Top1 down-regulation. In contrast to the effect of proteasome inhibition, inactivation of PARP1 was shown to increase the amount of CPT-induced SSBs and the level of ATM autophosphorylation. Together, these results support a model in which Top1 cleavage complexes arrest transcription and activate a ubiquitin-proteasome pathway leading to the degradation of Top1 cleavage complexes. Degradation of Top1 cleavage complexes results in the exposure of Top1-concealed SSBs for repair through a PARP1-dependent process. PMID:18515798

Polo-like kinase 1 inhibits DNA damage response during mitosis

PubMed Central

Benada, Jan; Burdová, Kamila; Lidak, Tomáš; von Morgen, Patrick; Macurek, Libor

2015-01-01

In response to genotoxic stress, cells protect their genome integrity by activation of a conserved DNA damage response (DDR) pathway that coordinates DNA repair and progression through the cell cycle. Extensive modification of the chromatin flanking the DNA lesion by ATM kinase and RNF8/RNF168 ubiquitin ligases enables recruitment of various repair factors. Among them BRCA1 and 53BP1 are required for homologous recombination and non-homologous end joining, respectively. Whereas mechanisms of DDR are relatively well understood in interphase cells, comparatively less is known about organization of DDR during mitosis. Although ATM can be activated in mitotic cells, 53BP1 is not recruited to the chromatin until cells exit mitosis. Here we report mitotic phosphorylation of 53BP1 by Plk1 and Cdk1 that impairs the ability of 53BP1 to bind the ubiquitinated H2A and to properly localize to the sites of DNA damage. Phosphorylation of 53BP1 at S1618 occurs at kinetochores and in cytosol and is restricted to mitotic cells. Interaction between 53BP1 and Plk1 depends on the activity of Cdk1. We propose that activity of Cdk1 and Plk1 allows spatiotemporally controlled suppression of 53BP1 function during mitosis. PMID:25607646

Polo-like kinase 1 inhibits DNA damage response during mitosis.

PubMed

Benada, Jan; Burdová, Kamila; Lidak, Tomáš; von Morgen, Patrick; Macurek, Libor

2015-01-01

In response to genotoxic stress, cells protect their genome integrity by activation of a conserved DNA damage response (DDR) pathway that coordinates DNA repair and progression through the cell cycle. Extensive modification of the chromatin flanking the DNA lesion by ATM kinase and RNF8/RNF168 ubiquitin ligases enables recruitment of various repair factors. Among them BRCA1 and 53BP1 are required for homologous recombination and non-homologous end joining, respectively. Whereas mechanisms of DDR are relatively well understood in interphase cells, comparatively less is known about organization of DDR during mitosis. Although ATM can be activated in mitotic cells, 53BP1 is not recruited to the chromatin until cells exit mitosis. Here we report mitotic phosphorylation of 53BP1 by Plk1 and Cdk1 that impairs the ability of 53BP1 to bind the ubiquitinated H2A and to properly localize to the sites of DNA damage. Phosphorylation of 53BP1 at S1618 occurs at kinetochores and in cytosol and is restricted to mitotic cells. Interaction between 53BP1 and Plk1 depends on the activity of Cdk1. We propose that activity of Cdk1 and Plk1 allows spatiotemporally controlled suppression of 53BP1 function during mitosis.

Ubiquitylation-dependent regulation of NEIL1 by Mule and TRIM26 is required for the cellular DNA damage response.

PubMed

Edmonds, Matthew J; Carter, Rachel J; Nickson, Catherine M; Williams, Sarah C; Parsons, Jason L

2017-01-25

Endonuclease VIII-like protein 1 (NEIL1) is a DNA glycosylase involved in initiating the base excision repair pathway, the major cellular mechanism for repairing DNA base damage. Here, we have purified the major E3 ubiquitin ligases from human cells responsible for regulation of NEIL1 by ubiquitylation. Interestingly, we have identified two enzymes that catalyse NEIL1 polyubiquitylation, Mcl-1 ubiquitin ligase E3 (Mule) and tripartite motif 26 (TRIM26). We demonstrate that these enzymes are capable of polyubiquitylating NEIL1 in vitro, and that both catalyse ubiquitylation of NEIL1 within the same C-terminal lysine residues. An siRNA-mediated knockdown of Mule or TRIM26 leads to stabilisation of NEIL1, demonstrating that these enzymes are important in regulating cellular NEIL1 steady state protein levels. Similarly, a mutant NEIL1 protein lacking residues for ubiquitylation is more stable than the wild type protein in vivo We also demonstrate that cellular NEIL1 protein is induced in response to ionising radiation (IR), although this occurs specifically in a Mule-dependent manner. Finally we show that stabilisation of NEIL1, particularly following TRIM26 siRNA, contributes to cellular resistance to IR. This highlights the importance of Mule and TRIM26 in maintaining steady state levels of NEIL1, but also those required for the cellular DNA damage response. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

RING finger and WD repeat domain 3 (RFWD3) associates with replication protein A (RPA) and facilitates RPA-mediated DNA damage response.

PubMed

Liu, Shangfeng; Chu, Jessica; Yucer, Nur; Leng, Mei; Wang, Shih-Ya; Chen, Benjamin P C; Hittelman, Walter N; Wang, Yi

2011-06-24

DNA damage response is crucial for maintaining genomic integrity and preventing cancer by coordinating the activation of checkpoints and the repair of damaged DNA. Central to DNA damage response are the two checkpoint kinases ATM and ATR that phosphorylate a wide range of substrates. RING finger and WD repeat domain 3 (RFWD3) was initially identified as a substrate of ATM/ATR from a proteomic screen. Subsequent studies showed that RFWD3 is an E3 ubiquitin ligase that ubiquitinates p53 in vitro and positively regulates p53 levels in response to DNA damage. We report here that RFWD3 associates with replication protein A (RPA), a single-stranded DNA-binding protein that plays essential roles in DNA replication, recombination, and repair. Binding of RPA to single-stranded DNA (ssDNA), which is generated by DNA damage and repair, is essential for the recruitment of DNA repair factors to damaged sites and the activation of checkpoint signaling. We show that RFWD3 is physically associated with RPA and rapidly localizes to sites of DNA damage in a RPA-dependent manner. In vitro experiments suggest that the C terminus of RFWD3, which encompass the coiled-coil domain and the WD40 domain, is necessary for binding to RPA. Furthermore, DNA damage-induced phosphorylation of RPA and RFWD3 is dependent upon each other. Consequently, loss of RFWD3 results in the persistent foci of DNA damage marker γH2AX and the repair protein Rad51 in damaged cells. These findings suggest that RFWD3 is recruited to sites of DNA damage and facilitates RPA-mediated DNA damage signaling and repair.

Proliferating Cell Nuclear Antigen-dependent Rapid Recruitment of Cdt1 and CRL4Cdt2 at DNA-damaged Sites after UV Irradiation in HeLa Cells*

PubMed Central

Ishii, Takashi; Shiomi, Yasushi; Takami, Toshihiro; Murakami, Yusuke; Ohnishi, Naho; Nishitani, Hideo

2010-01-01

The licensing factor Cdt1 is degraded by CRL4Cdt2 ubiquitin ligase dependent on proliferating cell nuclear antigen (PCNA) during S phase and when DNA damage is induced in G1 phase. Association of both Cdt2 and PCNA with chromatin was observed in S phase and after UV irradiation. Here we used a micropore UV irradiation assay to examine Cdt2 accumulation at cyclobutane pyrimidine dimer-containing DNA-damaged sites in the process of Cdt1 degradation in HeLa cells. Cdt2, present in the nucleus throughout the cell cycle, accumulated rapidly at damaged DNA sites during G1 phase. The recruitment of Cdt2 is dependent on prior PCNA chromatin binding because Cdt2 association was prevented when PCNA was silenced. Cdt1 was also recruited to damaged sites soon after UV irradiation through its PIP-box. As Cdt1 was degraded, the Cdt2 signal at damaged sites was reduced, but PCNA, cyclobutane pyrimidine dimer, and XPA (xeroderma pigmentosum, complementation group A) signals remained at the same levels. These findings suggest that Cdt1 degradation following UV irradiation occurs rapidly at damaged sites due to PCNA chromatin loading and the recruitment of Cdt1 and CRL4Cdt2, before DNA damage repair is completed. PMID:20929861

Ubiquitin-specific Protease 11 (USP11) Deubiquitinates Hybrid Small Ubiquitin-like Modifier (SUMO)-Ubiquitin Chains to Counteract RING Finger Protein 4 (RNF4)*

PubMed Central

Hendriks, Ivo A.; Schimmel, Joost; Eifler, Karolin; Olsen, Jesper V.; Vertegaal, Alfred C. O.

2015-01-01

Ring finger protein 4 (RNF4) is a SUMO-targeted ubiquitin E3 ligase with a pivotal function in the DNA damage response (DDR). SUMO interaction motifs (SIMs) in the N-terminal part of RNF4 tightly bind to SUMO polymers, and RNF4 can ubiquitinate these polymers in vitro. Using a proteomic approach, we identified the deubiquitinating enzyme ubiquitin-specific protease 11 (USP11), a known DDR-component, as a functional interactor of RNF4. USP11 can deubiquitinate hybrid SUMO-ubiquitin chains to counteract RNF4. SUMO-enriched nuclear bodies are stabilized by USP11, which functions downstream of RNF4 as a counterbalancing factor. In response to DNA damage induced by methyl methanesulfonate, USP11 could counteract RNF4 to inhibit the dissolution of nuclear bodies. Thus, we provide novel insight into cross-talk between ubiquitin and SUMO and uncover USP11 and RNF4 as a balanced SUMO-targeted ubiquitin ligase/protease pair with a role in the DDR. PMID:25969536

Nedd4 Family Interacting Protein 1 (Ndfip1) Is Required for Ubiquitination and Nuclear Trafficking of BRCA1-associated ATM Activator 1 (BRAT1) during the DNA Damage Response*

PubMed Central

Low, Ley-Hian; Chow, Yuh-Lit; Li, Yijia; Goh, Choo-Peng; Putz, Ulrich; Silke, John; Ouchi, Toru; Howitt, Jason; Tan, Seong-Seng

2015-01-01

During injury, cells are vulnerable to apoptosis from a variety of stress conditions including DNA damage causing double-stranded breaks. Without repair, these breaks lead to aberrations in DNA replication and transcription, leading to apoptosis. A major response to DNA damage is provided by the protein kinase ATM (ataxia telangiectasia mutated) that is capable of commanding a plethora of signaling networks for DNA repair, cell cycle arrest, and even apoptosis. A key element in the DNA damage response is the mobilization of activating proteins into the cell nucleus to repair damaged DNA. BRAT1 is one of these proteins, and it functions as an activator of ATM by maintaining its phosphorylated status while also keeping other phosphatases at bay. However, it is unknown how BRAT1 is trafficked into the cell nucleus to maintain ATM phosphorylation. Here we demonstrate that Ndfip1-mediated ubiquitination of BRAT1 leads to BRAT1 trafficking into the cell nucleus. Without Ndfip1, BRAT1 failed to translocate to the nucleus. Under genotoxic stress, cells showed increased expression of both Ndfip1 and phosphorylated ATM. Following brain injury, neurons show increased expression of Ndfip1 and nuclear translocation of BRAT1. These results point to Ndfip1 as a sensor protein during cell injury and Ndfip1 up-regulation as a cue for BRAT1 ubiquitination by Nedd4 E3 ligases, followed by nuclear translocation of BRAT1. PMID:25631046

Ubiquitination of exposed glycoproteins by SCFFBXO27 directs damaged lysosomes for autophagy

PubMed Central

Yoshida, Yukiko; Yasuda, Sayaka; Fujita, Toshiharu; Hamasaki, Maho; Murakami, Arisa; Kawawaki, Junko; Iwai, Kazuhiro; Saeki, Yasushi; Yoshimori, Tamotsu; Matsuda, Noriyuki; Tanaka, Keiji

2017-01-01

Ubiquitination functions as a signal to recruit autophagic machinery to damaged organelles and induce their clearance. Here, we report the characterization of FBXO27, a glycoprotein-specific F-box protein that is part of the SCF (SKP1/CUL1/F-box protein) ubiquitin ligase complex, and demonstrate that SCFFBXO27 ubiquitinates glycoproteins in damaged lysosomes to regulate autophagic machinery recruitment. Unlike F-box proteins in other SCF complexes, FBXO27 is subject to N-myristoylation, which localizes it to membranes, allowing it to accumulate rapidly around damaged lysosomes. We also screened for proteins that are ubiquitinated upon lysosomal damage, and identified two SNARE proteins, VAMP3 and VAMP7, and five lysosomal proteins, LAMP1, LAMP2, GNS, PSAP, and TMEM192. Ubiquitination of all glycoproteins identified in this screen increased upon FBXO27 overexpression. We found that the lysosomal protein LAMP2, which is ubiquitinated preferentially on lysosomal damage, enhances autophagic machinery recruitment to damaged lysosomes. Thus, we propose that SCFFBXO27 ubiquitinates glycoproteins exposed upon lysosomal damage to induce lysophagy. PMID:28743755

DNA-binding regulates site-specific ubiquitination of IRF-1.

PubMed

Landré, Vivien; Pion, Emmanuelle; Narayan, Vikram; Xirodimas, Dimitris P; Ball, Kathryn L

2013-02-01

Understanding the determinants for site-specific ubiquitination by E3 ligase components of the ubiquitin machinery is proving to be a challenge. In the present study we investigate the role of an E3 ligase docking site (Mf2 domain) in an intrinsically disordered domain of IRF-1 [IFN (interferon) regulatory factor-1], a short-lived IFNγ-regulated transcription factor, in ubiquitination of the protein. Ubiquitin modification of full-length IRF-1 by E3 ligases such as CHIP [C-terminus of the Hsc (heat-shock cognate) 70-interacting protein] and MDM2 (murine double minute 2), which dock to the Mf2 domain, was specific for lysine residues found predominantly in loop structures that extend from the DNA-binding domain, whereas no modification was detected in the more conformationally flexible C-terminal half of the protein. The E3 docking site was not available when IRF-1 was in its DNA-bound conformation and cognate DNA-binding sequences strongly suppressed ubiquitination, highlighting a strict relationship between ligase binding and site-specific modification at residues in the DNA-binding domain. Hyperubiquitination of a non-DNA-binding mutant supports a mechanism where an active DNA-bound pool of IRF-1 is protected from polyubiquitination and degradation.

Regulation of transcriptional activators by DNA-binding domain ubiquitination

PubMed Central

Landré, Vivien; Revi, Bhindu; Mir, Maria Gil; Verma, Chandra; Hupp, Ted R; Gilbert, Nick; Ball, Kathryn L

2017-01-01

Ubiquitin is a key component of the regulatory network that maintains gene expression in eukaryotes, yet the molecular mechanism(s) by which non-degradative ubiquitination modulates transcriptional activator (TA) function is unknown. Here endogenous p53, a stress-activated transcription factor required to maintain health, is stably monoubiquitinated, following pathway activation by IR or Nutlin-3 and localized to the nucleus where it becomes tightly associated with chromatin. Comparative structure–function analysis and in silico modelling demonstrate a direct role for DNA-binding domain (DBD) monoubiquitination in TA activation. When attached to the DBD of either p53, or a second TA IRF-1, ubiquitin is orientated towards, and makes contact with, the DNA. The contact is made between a predominantly cationic surface on ubiquitin and the anionic DNA. Our data demonstrate an unexpected role for ubiquitin in the mechanism of TA-activity enhancement and provides insight into a new level of transcriptional regulation. PMID:28362432

Spectrum of complex DNA damages depends on the incident radiation

NASA Astrophysics Data System (ADS)

Hada, M.; Sutherland, B.

Ionizing radiation induces clustered DNA damages in DNA-two or more abasic sites oxidized bases and strand breaks on opposite DNA strands within a few helical turns Clustered damages are considered to be difficult to repair and therefore potentially lethal and mutagenic damages Although induction of single strand breaks and isolated lesions has been studied extensively little is known of factors affecting induction of clusters other than double strand breaks DSB The aim of the present study was to determine whether the type of incident radiation could affect yield or spectra of specific clusters Genomic T7 DNA a simple 40 kbp linear blunt-ended molecule was irradiated in non-scavenging buffer conditions with Fe 970 MeV n Ti 980 MeV n C 293 MeV n Si 586 MeV n ions or protons 1 GeV n at the NASA Space Radiation Laboratory or with 100 kVp X-rays Irradiated DNA was treated with homogeneous Fpg or Nfo proteins or without enzyme treatment for DSB quantitation then electrophoresed in neutral agarose gels DSB Fpg-OxyPurine clusters and Nfo-Abasic clusters were quantified by number average length analysis The results show that the yields of all these complex damages depend on the incident radiation Although LETs are similar protons induced twice as many DSBs than did X-rays Further the spectrum of damage also depends on the radiation The yield damage Mbp Gy of all damages decreased with increasing linear energy transfer LET of the radiation The relative frequencies of DSBs to Abasic- and OxyBase clusters were higher

DNA-damage-inducible 1 protein (Ddi1) contains an uncharacteristic ubiquitin-like domain that binds ubiquitin

PubMed Central

Nowicka, Urszula; Zhang, Daoning; Walker, Olivier; Krutauz, Daria; Castañeda, Carlos A.; Chaturvedi, Apurva; Chen, Tony Y.; Reis, Noa; Glickman, Michael H.; Fushman, David

2015-01-01

SUMMARY Ddi1 belongs to a family of shuttle proteins targeting polyubiquitinated substrates for proteasomal degradation. Unlike the other proteasomal shuttles, Rad23 and Dsk2, Ddi1 remains an enigma: its function is not fully understood and structural properties are poorly characterized. We determined the structure and binding properties of the ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains of Ddi1 from Saccharomyces cerevisiae. We found that, while Ddi1UBA forms a characteristic UBA:ubiquitin complex, Ddi1UBL has entirely uncharacteristic binding preferences. Despite having a ubiquitin-like fold, Ddi1UBL does not interact with typical UBL-receptors but, unexpectedly, binds ubiquitin, forming a unique interface mediated by hydrophobic contacts and by salt-bridges between oppositely-charged residues of Ddi1UBL and ubiquitin. In stark contrast with ubiquitin and other UBLs, the β-sheet surface of Ddi1UBL is negatively charged and, therefore, is recognized in a completely different way. The dual functionality of Ddi1UBL, capable of binding both ubiquitin and proteasome, suggests a novel mechanism for Ddi1 as a proteasomal shuttle. PMID:25703377

Regulation of human MutYH DNA glycosylase by the E3 ubiquitin ligase mule.

PubMed

Dorn, Julia; Ferrari, Elena; Imhof, Ralph; Ziegler, Nathalie; Hübscher, Ulrich

2014-03-07

Oxidation of DNA is a frequent and constantly occurring event. One of the best characterized oxidative DNA lesions is 7,8-dihydro-8-oxoguanine (8-oxo-G). It instructs most DNA polymerases to preferentially insert an adenine (A) opposite 8-oxo-G instead of the appropriate cytosine (C) thus showing miscoding potential. The MutY DNA glycosylase homologue (MutYH) recognizes A:8-oxo-G mispairs and removes the mispaired A giving way to the canonical base excision repair that ultimately restores undamaged guanine (G). Here we characterize for the first time in detail a posttranslational modification of the human MutYH DNA glycosylase. We show that MutYH is ubiquitinated in vitro and in vivo by the E3 ligase Mule between amino acids 475 and 535. Mutation of five lysine residues in this region significantly stabilizes MutYH, suggesting that these are the target sites for ubiquitination. The endogenous MutYH protein levels depend on the amount of expressed Mule. Furthermore, MutYH and Mule physically interact. We found that a ubiquitination-deficient MutYH mutant shows enhanced binding to chromatin. The mutation frequency of the ovarian cancer cell line A2780, analyzed at the HPRT locus can be increased upon oxidative stress and depends on the MutYH levels that are regulated by Mule. This reflects the importance of tightly regulated MutYH levels in the cell. In summary our data show that ubiquitination is an important regulatory mechanism for the essential MutYH DNA glycosylase in human cells.

Disruption of DNA repair in cancer cells by ubiquitination of a destabilising dimerization domain of nucleotide excision repair protein ERCC1

PubMed Central

Yang, Lanlan; Ritchie, Ann-Marie; Melton, David W.

2017-01-01

DNA repair pathways present in all cells serve to preserve genome stability, but in cancer cells they also act reduce the efficacy of chemotherapy. The endonuclease ERCC1-XPF has an important role in the repair of DNA damage caused by a variety of chemotherapeutic agents and there has been intense interest in the use of ERCC1 as a predictive marker of therapeutic response in non-small cell lung carcinoma, squamous cell carcinoma and ovarian cancer. We have previously validated ERCC1 as a therapeutic target in melanoma, but all small molecule ERCC1-XPF inhibitors reported to date have lacked sufficient potency and specificity for clinical use. In an alternative approach to prevent the repair activity of ERCC1-XPF, we investigated the mechanism of ERCC1 ubiquitination and found that the key region was the C-terminal (HhH)2 domain which heterodimerizes with XPF. This ERCC1 region was modified by non-conventional lysine-independent, but proteasome-dependent polyubiquitination, involving Lys33 of ubiquitin and a linear ubiquitin chain. XPF was not polyubiquitinated and its expression was dependent on presence of ERCC1, but not vice versa. To our surprise we found that ERCC1 can also homodimerize through its C-terminal (HhH)2 domain. We exploited the ability of a peptide containing this C-terminal domain to destabilise both endogenous ERCC1 and XPF in human melanoma cells and fibroblasts, resulting in reductions of up to 85% in nucleotide excision repair and near two-fold increased sensitivity to DNA damaging agents. We suggest that the ERCC1 (HhH)2 domain could be used in an alternative strategy to treat cancer. PMID:28903417

DNA damage tolerance pathway involving DNA polymerase ι and the tumor suppressor p53 regulates DNA replication fork progression.

PubMed

Hampp, Stephanie; Kiessling, Tina; Buechle, Kerstin; Mansilla, Sabrina F; Thomale, Jürgen; Rall, Melanie; Ahn, Jinwoo; Pospiech, Helmut; Gottifredi, Vanesa; Wiesmüller, Lisa

2016-07-26

DNA damage tolerance facilitates the progression of replication forks that have encountered obstacles on the template strands. It involves either translesion DNA synthesis initiated by proliferating cell nuclear antigen monoubiquitination or less well-characterized fork reversal and template switch mechanisms. Herein, we characterize a novel tolerance pathway requiring the tumor suppressor p53, the translesion polymerase ι (POLι), the ubiquitin ligase Rad5-related helicase-like transcription factor (HLTF), and the SWI/SNF catalytic subunit (SNF2) translocase zinc finger ran-binding domain containing 3 (ZRANB3). This novel p53 activity is lost in the exonuclease-deficient but transcriptionally active p53(H115N) mutant. Wild-type p53, but not p53(H115N), associates with POLι in vivo. Strikingly, the concerted action of p53 and POLι decelerates nascent DNA elongation and promotes HLTF/ZRANB3-dependent recombination during unperturbed DNA replication. Particularly after cross-linker-induced replication stress, p53 and POLι also act together to promote meiotic recombination enzyme 11 (MRE11)-dependent accumulation of (phospho-)replication protein A (RPA)-coated ssDNA. These results implicate a direct role of p53 in the processing of replication forks encountering obstacles on the template strand. Our findings define an unprecedented function of p53 and POLι in the DNA damage response to endogenous or exogenous replication stress.

Intrinsic Flexibility of Ubiquitin on Proliferating Cell Nuclear Antigen (PCNA) in Translesion Synthesis*

PubMed Central

Hibbert, Richard G.; Sixma, Titia K.

2012-01-01

Ubiquitin conjugation provides a crucial signaling role in hundreds of cellular pathways; however, a structural understanding of ubiquitinated substrates is lacking. One important substrate is monoubiquitinated PCNA (PCNA-Ub), which signals for recruitment of damage-tolerant polymerases in the translesion synthesis (TLS) pathway of DNA damage avoidance. We use a novel and efficient enzymatic method to produce PCNA-Ub at high yield with a native isopeptide bond and study its Usp1/UAF1-dependent deconjugation. In solution we find that the ubiquitin moiety is flexible relative to the PCNA, with its hydrophobic patch mostly accessible for recruitment of TLS polymerases, which promotes the interaction with polymerase η. The studies are a prototype for the nature of the ubiquitin modification. PMID:22989887

Structural Basis for the Interaction of Mutasome Assembly Factor REV1 with Ubiquitin.

PubMed

Cui, Gaofeng; Botuyan, Maria Victoria; Mer, Georges

2018-05-18

REV1 is an evolutionarily conserved translesion synthesis (TLS) DNA polymerase and an assembly factor key for the recruitment of other TLS polymerases to DNA damage sites. REV1-mediated recognition of ubiquitin in the proliferative cell nuclear antigen is thought to be the trigger for TLS activation. Here we report the solution NMR structure of a 108-residue fragment of human REV1 encompassing the two putative ubiquitin-binding motifs UBM1 and UBM2 in complex with ubiquitin. While in mammals UBM1 and UBM2 are both required for optimal association of REV1 with replication factories after DNA damage, we show that only REV1 UBM2 binds ubiquitin. Structure-guided mutagenesis in Saccharomyces cerevisiae further highlights the importance of UBM2 for REV1-mediated mutagenesis and DNA damage tolerance. Copyright © 2018 Elsevier Ltd. All rights reserved.

The cAMP signaling system inhibits the repair of γ-ray-induced DNA damage by promoting Epac1-mediated proteasomal degradation of XRCC1 protein in human lung cancer cells.

PubMed

Cho, Eun-Ah; Juhnn, Yong-Sung

2012-06-01

Cyclic AMP is involved in the regulation of metabolism, gene expression, cellular growth and proliferation. Recently, the cAMP signaling system was found to modulate DNA-damaging agent-induced apoptosis by regulating the expression of Bcl-2 family proteins and inhibitors of apoptosis. Thus, we hypothesized that the cAMP signaling may modulate DNA repair activity, and we investigated the effects of the cAMP signaling system on γ-ray-induced DNA damage repair in lung cancer cells. Transient expression of a constitutively active mutant of stimulatory G protein (GαsQL) or treatment with forskolin, an adenylyl cyclase activator, augmented radiation-induced DNA damage and inhibited repair of the damage in H1299 lung cancer cells. Expression of GαsQL or treatment with forskolin or isoproterenol inhibited the radiation-induced expression of the XRCC1 protein, and exogenous expression of XRCC1 abolished the DNA repair-inhibiting effect of forskolin. Forskolin treatment promoted the ubiquitin and proteasome-dependent degradation of the XRCC1 protein, resulting in a significant decrease in the half-life of the protein after γ-ray irradiation. The effect of forskolin on XRCC1 expression was not inhibited by PKA inhibitor, but 8-pCPT-2'-O-Me-cAMP, an Epac-selective cAMP analog, increased ubiquitination of XRCC1 protein and decreased XRCC1 expression. Knockdown of Epac1 abolished the effect of 8-pCPT-2'-O-Me-cAMP and restored XRCC1 protein level following γ-ray irradiation. From these results, we conclude that the cAMP signaling system inhibits the repair of γ-ray-induced DNA damage by promoting the ubiquitin-proteasome dependent degradation of XRCC1 in an Epac-dependent pathway in lung cancer cells. Copyright © 2012 Elsevier Inc. All rights reserved.

A selective USP1-UAF1 inhibitor links deubiquitination to DNA damage responses

PubMed Central

Liang, Qin; Dexheimer, Thomas S; Zhang, Ping; Rosenthal, Andrew S; Villamil, Mark A; You, Changjun; Zhang, Qiuting; Chen, Junjun; Ott, Christine A; Sun, Hongmao; Luci, Diane K; Yuan, Bifeng; Simeonov, Anton; Jadhav, Ajit; Xiao, Hui; Wang, Yinsheng; Maloney, David J; Zhuang, Zhihao

2014-01-01

Protein ubiquitination and deubiquitination are central to the control of a large number of cellular pathways and signaling networks in eukaryotes. Although the essential roles of ubiquitination have been established in the eukaryotic DNA damage response, the deubiquitination process remains poorly defined. Chemical probes that perturb the activity of deubiquitinases (DUBs) are needed to characterize the cellular function of deubiquitination. Here we report ML323 (2), a highly potent inhibitor of the USP1-UAF1 deubiquitinase complex with excellent selectivity against human DUBs, deSUMOylase, deneddylase and unrelated proteases. Using ML323, we interrogated deubiquitination in the cellular response to UV- and cisplatin-induced DNA damage and revealed new insights into the requirement of deubiquitination in the DNA translesion synthesis and Fanconi anemia pathways. Moreover, ML323 potentiates cisplatin cytotoxicity in non-small cell lung cancer and osteosarcoma cells. Our findings point to USP1-UAF1 as a key regulator of the DNA damage response and a target for overcoming resistance to the platinum-based anticancer drugs. PMID:24531842

How Chemical Synthesis of Ubiquitin Conjugates Helps To Understand Ubiquitin Signal Transduction.

PubMed

Hameed, Dharjath S; Sapmaz, Aysegul; Ovaa, Huib

2017-03-15

Ubiquitin (Ub) is a small post-translational modifier protein involved in a myriad of biochemical processes including DNA damage repair, proteasomal proteolysis, and cell cycle control. Ubiquitin signaling pathways have not been completely deciphered due to the complex nature of the enzymes involved in ubiquitin conjugation and deconjugation. Hence, probes and assay reagents are important to get a better understanding of this pathway. Recently, improvements have been made in synthesis procedures of Ub derivatives. In this perspective, we explain various research reagents available and how chemical synthesis has made an important contribution to Ub research.

Bioenergetic metabolites regulate base excision repair dependent cell death in response to DNA damage

PubMed Central

Tang, Jiang-bo; Goellner, Eva M.; Wang, Xiao-hong; Trivedi, Ram N.; Croix, Claudette M. St; Jelezcova, Elena; Svilar, David; Brown, Ashley R.; Sobol, Robert W.

2009-01-01

Base excision repair (BER) protein expression is important for resistance to DNA damage-induced cytotoxicity. Conversely, BER imbalance (Polß deficiency or repair inhibition) enhances cytotoxicity of radiation and chemotherapeutic DNA-damaging agents. Whereas inhibition of critical steps in the BER pathway result in the accumulation of cytotoxic DNA double-strand breaks, we report that DNA damage-induced cytotoxicity due to deficiency in the BER protein Polß triggers cell death dependent on PARP activation yet independent of poly(ADP-ribose) (PAR)-mediated AIF nuclear translocation or PARG, suggesting that cytotoxicity is not from PAR or PAR-catabolite signaling. Cell death is rescued by the NAD+ metabolite NMN and is synergistic with inhibition of NAD+ biosynthesis, demonstrating that DNA damage-induced cytotoxicity mediated via BER inhibition is primarily dependent on cellular metabolite bioavailability. We offer a mechanistic justification for the elevated alkylation-induced cytotoxicity of Polß deficient cells, suggesting a linkage between DNA repair, cell survival and cellular bioenergetics. PMID:20068071

Regulation of Nucleotide Excision Repair by UV-DDB: Prioritization of Damage Recognition to Internucleosomal DNA

PubMed Central

Luch, Andreas; Glas, Andreas; Carell, Thomas; Naegeli, Hanspeter

2011-01-01

How tightly packed chromatin is thoroughly inspected for DNA damage is one of the fundamental unanswered questions in biology. In particular, the effective excision of carcinogenic lesions caused by the ultraviolet (UV) radiation of sunlight depends on UV-damaged DNA-binding protein (UV-DDB), but the mechanism by which this DDB1-DDB2 heterodimer stimulates DNA repair remained enigmatic. We hypothesized that a distinctive function of this unique sensor is to coordinate damage recognition in the nucleosome repeat landscape of chromatin. Therefore, the nucleosomes of human cells have been dissected by micrococcal nuclease, thus revealing, to our knowledge for the first time, that UV-DDB associates preferentially with lesions in hypersensitive, hence, highly accessible internucleosomal sites joining the core particles. Surprisingly, the accompanying CUL4A ubiquitin ligase activity is necessary to retain the xeroderma pigmentosum group C (XPC) partner at such internucleosomal repair hotspots that undergo very fast excision kinetics. This CUL4A complex thereby counteracts an unexpected affinity of XPC for core particles that are less permissive than hypersensitive sites to downstream repair subunits. That UV-DDB also adopts a ubiquitin-independent function is evidenced by domain mapping and in situ protein dynamics studies, revealing direct but transient interactions that promote a thermodynamically unfavorable β-hairpin insertion of XPC into substrate DNA. We conclude that the evolutionary advent of UV-DDB correlates with the need for a spatiotemporal organizer of XPC positioning in higher eukaryotic chromatin. PMID:22039351

53BP1 is a reader of the DNA damage-induced H2A Lys15 ubiquitin mark

PubMed Central

Fradet-Turcotte, Amélie; Canny, Marella D.; Escribano-Díaz, Cristina; Orthwein, Alexandre; Leung, Charles C.Y.; Huang, Hao; Landry, Marie-Claude; Kitevski-LeBlanc, Julianne; Noordermeer, Sylvie M.; Sicheri, Frank; Durocher, Daniel

2014-01-01

53BP1 (TP53BP1) is a chromatin-associated factor that promotes immunoglobulin class switching and DNA double-strand break (DSB) repair by non-homologous end joining. To accomplish its function in DNA repair, 53BP1 accumulates at DSB sites downstream of the RNF168 ubiquitin ligase. How ubiquitin recruits 53BP1 to break sites remains enigmatic since its relocalization involves recognition of H4 Lys20 (H4K20) methylation by its Tudor domain. Here we elucidate how 53BP1 is recruited to the chromatin that flanks DSB sites. We show that 53BP1 recognizes mono-nucleosomes containing dimethylated H4K20 (H4K20me2) and H2A ubiquitylated on Lys15 (H2AK15ub), the latter being a product of RNF168 action on chromatin. 53BP1 binds to nucleosomes minimally as a dimer using its previously characterized methyl-lysine-binding Tudor domain and a C-terminal extension, termed the ubiquitylation-dependent recruitment (UDR) motif, which interacts with the epitope formed by H2AK15ub and its surrounding residues on the H2A tail. 53BP1 is therefore a bivalent histone modification reader that recognizes a histone “code” produced by DSB signaling. PMID:23760478

Interplay between Ubiquitin, SUMO, and Poly(ADP-Ribose) in the Cellular Response to Genotoxic Stress

PubMed Central

Pellegrino, Stefania; Altmeyer, Matthias

2016-01-01

Cells employ a complex network of molecular pathways to cope with endogenous and exogenous genotoxic stress. This multilayered response ensures that genomic lesions are efficiently detected and faithfully repaired in order to safeguard genome integrity. The molecular choreography at sites of DNA damage relies heavily on post-translational modifications (PTMs). Protein modifications with ubiquitin and the small ubiquitin-like modifier SUMO have recently emerged as important regulatory means to coordinate DNA damage signaling and repair. Both ubiquitylation and SUMOylation can lead to extensive chain-like protein modifications, a feature that is shared with yet another DNA damage-induced PTM, the modification of proteins with poly(ADP-ribose) (PAR). Chains of ubiquitin, SUMO, and PAR all contribute to the multi-protein assemblies found at sites of DNA damage and regulate their spatio-temporal dynamics. Here, we review recent advancements in our understanding of how ubiquitin, SUMO, and PAR coordinate the DNA damage response and highlight emerging examples of an intricate interplay between these chain-like modifications during the cellular response to genotoxic stress. PMID:27148359

RFWD3-Mediated Ubiquitination Promotes Timely Removal of Both RPA and RAD51 from DNA Damage Sites to Facilitate Homologous Recombination.

PubMed

Inano, Shojiro; Sato, Koichi; Katsuki, Yoko; Kobayashi, Wataru; Tanaka, Hiroki; Nakajima, Kazuhiro; Nakada, Shinichiro; Miyoshi, Hiroyuki; Knies, Kerstin; Takaori-Kondo, Akifumi; Schindler, Detlev; Ishiai, Masamichi; Kurumizaka, Hitoshi; Takata, Minoru

2017-06-01

RFWD3 is a recently identified Fanconi anemia protein FANCW whose E3 ligase activity toward RPA is essential in homologous recombination (HR) repair. However, how RPA ubiquitination promotes HR remained unknown. Here, we identified RAD51, the central HR protein, as another target of RFWD3. We show that RFWD3 polyubiquitinates both RPA and RAD51 in vitro and in vivo. Phosphorylation by ATR and ATM kinases is required for this activity in vivo. RFWD3 inhibits persistent mitomycin C (MMC)-induced RAD51 and RPA foci by promoting VCP/p97-mediated protein dynamics and subsequent degradation. Furthermore, MMC-induced chromatin loading of MCM8 and RAD54 is defective in cells with inactivated RFWD3 or expressing a ubiquitination-deficient mutant RAD51. Collectively, our data reveal a mechanism that facilitates timely removal of RPA and RAD51 from DNA damage sites, which is crucial for progression to the late-phase HR and suppression of the FA phenotype. Copyright © 2017 Elsevier Inc. All rights reserved.

Controlling the response to DNA damage by the APC/C-Cdh1.

PubMed

de Boer, H Rudolf; Guerrero Llobet, S; van Vugt, Marcel A T M

2016-03-01

Proper cell cycle progression is safeguarded by the oscillating activities of cyclin/cyclin-dependent kinase complexes. An important player in the regulation of mitotic cyclins is the anaphase-promoting complex/cyclosome (APC/C), a multi-subunit E3 ubiquitin ligase. Prior to entry into mitosis, the APC/C remains inactive, which allows the accumulation of mitotic regulators. APC/C activation requires binding to either the Cdc20 or Cdh1 adaptor protein, which sequentially bind the APC/C and facilitate targeting of multiple mitotic regulators for proteasomal destruction, including Securin and Cyclin B, to ensure proper chromosome segregation and mitotic exit. Emerging data have indicated that the APC/C, particularly in association with Cdh1, also functions prior to mitotic entry. Specifically, the APC/C-Cdh1 is activated in response to DNA damage in G2 phase cells. These observations are in line with in vitro and in vivo genetic studies, in which cells lacking Cdh1 expression display various defects, including impaired DNA repair and aberrant cell cycle checkpoints. In this review, we summarize the current literature on APC/C regulation in response to DNA damage, the functions of APC/C-Cdh1 activation upon DNA damage, and speculate how APC/C-Cdh1 can control cell fate in the context of persistent DNA damage.

ssDNA damage dependence from singlet oxygen concentration at photodynamic interaction

NASA Astrophysics Data System (ADS)

Klimenko, V. V.; Kaydanov, N. E.; Emelyanov, A. K.; Bogdanov, A. A.

2017-11-01

Single stranded DNA damage at photodynamic treatment with Radachlorin photosensitizer was investigated. Chemical trap method was used to evaluate generation of singlet oxygen in water solution. Interaction of singlet oxygen with ssDNA resulted into decrease of the replication activity of ssDNA. DNA stopped replicating during PCR at irradiation doses greater than 15 J/cm2 and concentration of photosensitizer [PS] = 3.8 μM. The dependence of replication activity of ssDNA on generated singlet oxygen concentration was identified.

ATM-dependent pathways of chromatin remodelling and oxidative DNA damage responses.

PubMed

Berger, N Daniel; Stanley, Fintan K T; Moore, Shaun; Goodarzi, Aaron A

2017-10-05

Ataxia-telangiectasia mutated (ATM) is a serine/threonine protein kinase with a master regulatory function in the DNA damage response. In this role, ATM commands a complex biochemical network that signals the presence of oxidative DNA damage, including the dangerous DNA double-strand break, and facilitates subsequent repair. Here, we review the current state of knowledge regarding ATM-dependent chromatin remodelling and epigenomic alterations that are required to maintain genomic integrity in the presence of DNA double-strand breaks and/or oxidative stress. We will focus particularly on the roles of ATM in adjusting nucleosome spacing at sites of unresolved DNA double-strand breaks within complex chromatin environments, and the impact of ATM on preserving the health of cells within the mammalian central nervous system.This article is part of the themed issue 'Chromatin modifiers and remodellers in DNA repair and signalling'. © 2017 The Author(s).

New orally active DNA minor groove binding small molecule CT-1 acts against breast cancer by targeting tumor DNA damage leading to p53-dependent apoptosis.

PubMed

Saini, Karan Singh; Hamidullah; Ashraf, Raghib; Mandalapu, Dhanaraju; Das, Sharmistha; Siddiqui, Mohd Quadir; Dwivedi, Sonam; Sarkar, Jayanta; Sharma, Vishnu Lal; Konwar, Rituraj

2017-04-01

Targeting tumor DNA damage and p53 pathway is a clinically established strategy in the development of cancer chemotherapeutics. Majority of anti-cancer drugs are delivered through parenteral route for reasons like severe toxicity, lack of stability, and poor enteral absorption. Current DNA targeting drugs in clinical like anthracycline suffers from major drawbacks like cardiotoxicity. Here, we report identification of a new orally active small molecule curcumin-triazole conjugate (CT-1) with significant anti-breast cancer activity in vitro and in vivo. CT-1 selectively and significantly inhibits viability of breast cancer cell lines; retards cells cycle progression at S phase and induce mitochondrial-mediated cell apoptosis. CT-1 selectively binds to minor groove of DNA and induces DNA damage leading to increase in p53 along with decrease in its ubiquitination. Inhibition of p53 with pharmacological inhibitor as well as siRNA revealed the necessity of p53 in CT-1-mediated anti-cancer effects in breast cancer cells. Studies using several other intact p53 and deficient p53 cancer cell lines further confirmed necessity of p53 in CT-1-mediated anti-cancer response. Pharmacological inhibition of pan-caspase showed CT-1 induces caspase-dependent cell death in breast cancer cells. Most interestingly, oral administration of CT-1 induces significant inhibition of tumor growth in LA-7 syngeneic orthotropic rat mammary tumor model. CT-1 treated mammary tumor shows enhancement in DNA damage, p53 upregulation, and apoptosis. Collectively, CT-1 exhibits potent anti-cancer effect both in vitro and in vivo and could serve as a safe orally active lead for anti-cancer drug development. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Put a RING on it: regulation and inhibition of RNF8 and RNF168 RING finger E3 ligases at DNA damage sites

PubMed Central

Bartocci, Cristina; Denchi, Eros Lazzerini

2013-01-01

RING (Really Interesting New Gene) domain-containing E3 ubiquitin ligases comprise a large family of enzymes that in combination with an E2 ubiquitin-conjugating enzyme, modify target proteins by attaching ubiquitin moieties. A number of RING E3s play an essential role in the cellular response to DNA damage highlighting a crucial contribution for ubiquitin-mediated signaling to the genome surveillance pathway. Among the RING E3s, RNF8 and RNF168 play a critical role in the response to double stranded breaks, one of the most deleterious types of DNA damage. These proteins act as positive regulators of the signaling cascade that initiates at DNA lesions. Inactivation of these enzymes is sufficient to severely impair the ability of cells to respond to DNA damage. Given their central role in the pathway, several layers of regulation act at this nodal signaling point. Here we will summarize current knowledge on the roles of RNF8 and RNF168 in maintaining genome integrity with particular emphasis on recent insights into the multiple layers of regulation that act on these enzymes to fine-tune the cellular response to DNA lesions. PMID:23847653

The cAMP signaling system inhibits the repair of {gamma}-ray-induced DNA damage by promoting Epac1-mediated proteasomal degradation of XRCC1 protein in human lung cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cho, Eun-Ah; Juhnn, Yong-Sung, E-mail: juhnn@snu.ac.kr

2012-06-01

Highlights: Black-Right-Pointing-Pointer cAMP signaling system inhibits repair of {gamma}-ray-induced DNA damage. Black-Right-Pointing-Pointer cAMP signaling system inhibits DNA damage repair by decreasing XRCC1 expression. Black-Right-Pointing-Pointer cAMP signaling system decreases XRCC1 expression by promoting its proteasomal degradation. Black-Right-Pointing-Pointer The promotion of XRCC1 degradation by cAMP signaling system is mediated by Epac1. -- Abstract: Cyclic AMP is involved in the regulation of metabolism, gene expression, cellular growth and proliferation. Recently, the cAMP signaling system was found to modulate DNA-damaging agent-induced apoptosis by regulating the expression of Bcl-2 family proteins and inhibitors of apoptosis. Thus, we hypothesized that the cAMP signaling may modulate DNAmore » repair activity, and we investigated the effects of the cAMP signaling system on {gamma}-ray-induced DNA damage repair in lung cancer cells. Transient expression of a constitutively active mutant of stimulatory G protein (G{alpha}sQL) or treatment with forskolin, an adenylyl cyclase activator, augmented radiation-induced DNA damage and inhibited repair of the damage in H1299 lung cancer cells. Expression of G{alpha}sQL or treatment with forskolin or isoproterenol inhibited the radiation-induced expression of the XRCC1 protein, and exogenous expression of XRCC1 abolished the DNA repair-inhibiting effect of forskolin. Forskolin treatment promoted the ubiquitin and proteasome-dependent degradation of the XRCC1 protein, resulting in a significant decrease in the half-life of the protein after {gamma}-ray irradiation. The effect of forskolin on XRCC1 expression was not inhibited by PKA inhibitor, but 8-pCPT-2 Prime -O-Me-cAMP, an Epac-selective cAMP analog, increased ubiquitination of XRCC1 protein and decreased XRCC1 expression. Knockdown of Epac1 abolished the effect of 8-pCPT-2 Prime -O-Me-cAMP and restored XRCC1 protein level following {gamma

Chk2 and REGγ-dependent DBC1 regulation in DNA damage induced apoptosis

PubMed Central

Magni, Martina; Ruscica, Vincenzo; Buscemi, Giacomo; Kim, Ja-Eun; Nachimuthu, Benjamin Tamilselvan; Fontanella, Enrico; Delia, Domenico; Zannini, Laura

2014-01-01

Human DBC1 (Deleted in Breast Cancer 1; KIAA1967; CCAR2) is a protein implicated in the regulation of apoptosis, transcription and histone modifications. Upon DNA damage, DBC1 is phosphorylated by ATM/ATR on Thr454 and this modification increases its inhibitory interaction with SIRT1, leading to p53 acetylation and p53-dependent apoptosis. Here, we report that the inhibition of SIRT1 by DBC1 in the DNA damage response (DDR) also depends on Chk2, the transducer kinase that is activated by ATM upon DNA lesions and contributes to the spreading of DNA damage signal. Indeed we found that inactivation of Chk2 reduces DBC1-SIRT1 binding, thus preventing p53 acetylation and DBC1-induced apoptosis. These events are mediated by Chk2 phosphorylation of the 11S proteasome activator REGγ on Ser247, which increases REGγ-DBC1 interaction and SIRT1 inhibition. Overall our results clarify the mechanisms underlying the DBC1-dependent SIRT1 inhibition and link, for the first time, Chk2 and REGγ to the ATM-DBC1-SIRT1 axis. PMID:25361978

Dynamic survey of mitochondria by ubiquitin

PubMed Central

Escobar-Henriques, Mafalda; Langer, Thomas

2014-01-01

Ubiquitin is a post-translational modifier with proteolytic and non-proteolytic roles in many biological processes. At mitochondria, it performs regulatory homeostatic functions and contributes to mitochondrial quality control. Ubiquitin is essential for mitochondrial fusion, regulates mitochondria-ER contacts, and participates in maternal mtDNA inheritance. Under stress, mitochondrial dysfunction induces ubiquitin-dependent responses that involve mitochondrial proteome remodeling and culminate in organelle removal by mitophagy. In addition, many ubiquitin-dependent mechanisms have been shown to regulate innate immune responses and xenophagy. Here, we review the emerging roles of ubiquitin at mitochondria. PMID:24569520

Ubiquitin facilitates a quality-control pathway that removes damaged chloroplasts

DOE PAGES

Woodson, Jesse D.; Joens, Matthew S.; Sinson, Andrew B.; ...

2015-10-23

Energy production by chloroplasts and mitochondria causes constant oxidative damage. A functioning photosynthetic cell requires quality-control mechanisms to turn over and degrade chloroplasts damaged by reactive oxygen species (ROS). Here in this study, we generated a conditionally lethal Arabidopsis mutant that accumulated excess protoporphyrin IX in the chloroplast and produced singlet oxygen. Damaged chloroplasts were subsequently ubiquitinated and selectively degraded. A genetic screen identified the plant U-box 4 (PUB4) E3 ubiquitin ligase as being necessary for this process. pub4-6 mutants had defects in stress adaptation and longevity. As a result, we have identified a signal that leads to the targetedmore » removal of ROS-overproducing chloroplasts.« less

Ubiquitin facilitates a quality-control pathway that removes damaged chloroplasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Woodson, Jesse D.; Joens, Matthew S.; Sinson, Andrew B.

Energy production by chloroplasts and mitochondria causes constant oxidative damage. A functioning photosynthetic cell requires quality-control mechanisms to turn over and degrade chloroplasts damaged by reactive oxygen species (ROS). Here in this study, we generated a conditionally lethal Arabidopsis mutant that accumulated excess protoporphyrin IX in the chloroplast and produced singlet oxygen. Damaged chloroplasts were subsequently ubiquitinated and selectively degraded. A genetic screen identified the plant U-box 4 (PUB4) E3 ubiquitin ligase as being necessary for this process. pub4-6 mutants had defects in stress adaptation and longevity. As a result, we have identified a signal that leads to the targetedmore » removal of ROS-overproducing chloroplasts.« less

Parkin-catalyzed Ubiquitin-Ester Transfer Is Triggered by PINK1-dependent Phosphorylation*

PubMed Central

Iguchi, Masahiro; Kujuro, Yuki; Okatsu, Kei; Koyano, Fumika; Kosako, Hidetaka; Kimura, Mayumi; Suzuki, Norihiro; Uchiyama, Shinichiro; Tanaka, Keiji; Matsuda, Noriyuki

2013-01-01

PINK1 and PARKIN are causal genes for autosomal recessive familial Parkinsonism. PINK1 is a mitochondrial Ser/Thr kinase, whereas Parkin functions as an E3 ubiquitin ligase. Under steady-state conditions, Parkin localizes to the cytoplasm where its E3 activity is repressed. A decrease in mitochondrial membrane potential triggers Parkin E3 activity and recruits it to depolarized mitochondria for ubiquitylation of mitochondrial substrates. The molecular basis for how the E3 activity of Parkin is re-established by mitochondrial damage has yet to be determined. Here we provide in vitro biochemical evidence for ubiquitin-thioester formation on Cys-431 of recombinant Parkin. We also report that Parkin forms a ubiquitin-ester following a decrease in mitochondrial membrane potential in cells, and that this event is essential for substrate ubiquitylation. Importantly, the Parkin RING2 domain acts as a transthiolation or acyl-transferring domain rather than an E2-recruiting domain. Furthermore, formation of the ubiquitin-ester depends on PINK1 phosphorylation of Parkin Ser-65. A phosphorylation-deficient mutation completely inhibited formation of the Parkin ubiquitin-ester intermediate, whereas phosphorylation mimics, such as Ser to Glu substitution, enabled partial formation of the intermediate irrespective of Ser-65 phosphorylation. We propose that PINK1-dependent phosphorylation of Parkin leads to the ubiquitin-ester transfer reaction of the RING2 domain, and that this is an essential step in Parkin activation. PMID:23754282

Fanconi anemia: a disorder defective in the DNA damage response.

PubMed

Kitao, Hiroyuki; Takata, Minoru

2011-04-01

Fanconi anemia (FA) is a cancer predisposition disorder characterized by progressive bone marrow failure, congenital developmental defects, chromosomal abnormalities, and cellular hypersensitivity to DNA interstrand crosslink (ICL) agents. So far mutations in 14 FANC genes were identified in FA or FA-like patients. These gene products constitute a common ubiquitin-phosphorylation network called the "FA pathway" and cooperate with other proteins involved in DNA repair and cell cycle control to repair ICL lesions and to maintain genome stability. In this review, we summarize recent exciting discoveries that have expanded our view of the molecular mechanisms operating in DNA repair and DNA damage signaling.

Checkpoint Kinase-Dependent Regulation of DNA Repair and Genome Instability in Breast Cancer

DTIC Science & Technology

2009-06-01

4A-mediated ubiquitination and deg- radation. J. Biol. Chem. 276:48175–48182. 14. Chu, G., and E. Chang. 1988. Xeroderma pigmentosum group E cells lack...a nuclear factor that binds to damaged DNA. Science 242:564–567. 15. Cleaver, J. E. 2005. Cancer in xeroderma pigmentosum and related disorders of...28. Hwang, B. J., J. M. Ford, P. C. Hanawalt, and G. Chu. 1999. Expression of the p48 xeroderma pigmentosum gene is p53-dependent and is involved in

Diseases Associated with Defective Responses to DNA Damage

PubMed Central

O’Driscoll, Mark

2012-01-01

Within the last decade, multiple novel congenital human disorders have been described with genetic defects in known and/or novel components of several well-known DNA repair and damage response pathways. Examples include disorders of impaired nucleotide excision repair, DNA double-strand and single-strand break repair, as well as compromised DNA damage-induced signal transduction including phosphorylation and ubiquitination. These conditions further reinforce the importance of multiple genome stability pathways for health and development in humans. Furthermore, these conditions inform our knowledge of the biology of the mechanics of genome stability and in some cases provide potential routes to help exploit these pathways therapeutically. Here, I will review a selection of these exciting findings from the perspective of the disorders themselves, describing how they were identified, how genotype informs phenotype, and how these defects contribute to our growing understanding of genome stability pathways. PMID:23209155

Curcumin-Mediated HDAC Inhibition Suppresses the DNA Damage Response and Contributes to Increased DNA Damage Sensitivity

PubMed Central

Wang, Shu-Huei; Lin, Pei-Ya; Chiu, Ya-Chen; Huang, Ju-Sui; Kuo, Yi-Tsen; Wu, Jen-Chine; Chen, Chin-Chuan

2015-01-01

Chemo- and radiotherapy cause multiple forms of DNA damage and lead to the death of cancer cells. Inhibitors of the DNA damage response are candidate drugs for use in combination therapies to increase the efficacy of such treatments. In this study, we show that curcumin, a plant polyphenol, sensitizes budding yeast to DNA damage by counteracting the DNA damage response. Following DNA damage, the Mec1-dependent DNA damage checkpoint is inactivated and Rad52 recombinase is degraded by curcumin, which results in deficiencies in double-stand break repair. Additive effects on damage-induced apoptosis and the inhibition of damage-induced autophagy by curcumin were observed. Moreover, rpd3 mutants were found to mimic the curcumin-induced suppression of the DNA damage response. In contrast, hat1 mutants were resistant to DNA damage, and Rad52 degradation was impaired following curcumin treatment. These results indicate that the histone deacetylase inhibitor activity of curcumin is critical to DSB repair and DNA damage sensitivity. PMID:26218133

PKC-Dependent GlyT1 Ubiquitination Occurs Independent of Phosphorylation: Inespecificity in Lysine Selection for Ubiquitination

PubMed Central

Barrera, Susana P.; Castrejon-Tellez, Vicente; Trinidad, Margarita; Robles-Escajeda, Elisa; Vargas-Medrano, Javier; Varela-Ramirez, Armando; Miranda, Manuel

2015-01-01

Neurotransmitter transporter ubiquitination is emerging as the main mechanism for endocytosis and sorting of cargo into lysosomes. In this study, we demonstrate PKC-dependent ubiquitination of three different isoforms of the glycine transporter 1 (GlyT1). Incubation of cells expressing transporter with the PKC activator phorbol ester induced a dramatic, time-dependent increase in GlyT1 ubiquitination, followed by accumulation of GlyT1 in EEA1 positive early endosomes. This occurred via a mechanism that was abolished by inhibition of PKC. GlyT1 endocytosis was confirmed in both retinal sections and primary cultures of mouse amacrine neurons. Replacement of only all lysines in the N-and C-termini to arginines prevented ubiquitination and endocytosis, displaying redundancy in the mechanism of ubiquitination. Interestingly, a 40–50% reduction in glycine uptake was detected in phorbol-ester stimulated cells expressing the WT-GlyT1, whereas no significant change was for the mutant protein, demonstrating that endocytosis participates in the reduction of uptake. Consistent with previous findings for the dopamine transporter DAT, ubiquitination of GlyT1 tails functions as sorting signal to deliver transporter into the lysosome and removal of ubiquitination sites dramatically attenuated the rate of GlyT1 degradation. Finally, we showed for the first time that PKC-dependent GlyT1 phosphorylation was not affected by removal of ubiquitination sites, suggesting separate PKC-dependent signaling events for these posttranslational modifications. PMID:26418248

Characterization of human translesion DNA synthesis across a UV-induced DNA lesion

PubMed Central

Hedglin, Mark; Pandey, Binod; Benkovic, Stephen J

2016-01-01

Translesion DNA synthesis (TLS) during S-phase uses specialized TLS DNA polymerases to replicate a DNA lesion, allowing stringent DNA synthesis to resume beyond the offending damage. Human TLS involves the conjugation of ubiquitin to PCNA clamps encircling damaged DNA and the role of this post-translational modification is under scrutiny. A widely-accepted model purports that ubiquitinated PCNA recruits TLS polymerases such as pol η to sites of DNA damage where they may also displace a blocked replicative polymerase. We provide extensive quantitative evidence that the binding of pol η to PCNA and the ensuing TLS are both independent of PCNA ubiquitination. Rather, the unique properties of pols η and δ are attuned to promote an efficient and passive exchange of polymerases during TLS on the lagging strand. DOI: http://dx.doi.org/10.7554/eLife.19788.001 PMID:27770570

The distribution of DNA damage is defined by region-specific susceptibility to DNA damage formation rather than repair differences.

PubMed

Strand, Janne M; Scheffler, Katja; Bjørås, Magnar; Eide, Lars

2014-06-01

The cellular genomes are continuously damaged by reactive oxygen species (ROS) from aerobic processes. The impact of DNA damage depends on the specific site as well as the cellular state. The steady-state level of DNA damage is the net result of continuous formation and subsequent repair, but it is unknown to what extent heterogeneous damage distribution is caused by variations in formation or repair of DNA damage. Here, we used a restriction enzyme/qPCR based method to analyze DNA damage in promoter and coding regions of four nuclear genes: the two house-keeping genes Gadph and Tbp, and the Ndufa9 and Ndufs2 genes encoding mitochondrial complex I subunits, as well as mt-Rnr1 encoded by mitochondrial DNA (mtDNA). The distribution of steady-state levels of damage varied in a site-specific manner. Oxidative stress induced damage in nDNA to a similar extent in promoter and coding regions, and more so in mtDNA. The subsequent removal of damage from nDNA was efficient and comparable with recovery times depending on the initial damage load, while repair of mtDNA was delayed with subsequently slower repair rate. The repair was furthermore found to be independent of transcription or the transcription-coupled repair factor CSB, but dependent on cellular ATP. Our results demonstrate that the capacity to repair DNA is sufficient to remove exogenously induced damage. Thus, we conclude that the heterogeneous steady-state level of DNA damage in promoters and coding regions is caused by site-specific DNA damage/modifications that take place under normal metabolism. Copyright © 2014 Elsevier B.V. All rights reserved.

DNA damage during S-phase mediates the proliferation-quiescence decision in the subsequent G1 via p21 expression

PubMed Central

Barr, Alexis R.; Cooper, Samuel; Heldt, Frank S.; Butera, Francesca; Stoy, Henriette; Mansfeld, Jörg; Novák, Béla; Bakal, Chris

2017-01-01

Following DNA damage caused by exogenous sources, such as ionizing radiation, the tumour suppressor p53 mediates cell cycle arrest via expression of the CDK inhibitor, p21. However, the role of p21 in maintaining genomic stability in the absence of exogenous DNA-damaging agents is unclear. Here, using live single-cell measurements of p21 protein in proliferating cultures, we show that naturally occurring DNA damage incurred over S-phase causes p53-dependent accumulation of p21 during mother G2- and daughter G1-phases. High p21 levels mediate G1 arrest via CDK inhibition, yet lower levels have no impact on G1 progression, and the ubiquitin ligases CRL4Cdt2 and SCFSkp2 couple to degrade p21 prior to the G1/S transition. Mathematical modelling reveals that a bistable switch, created by CRL4Cdt2, promotes irreversible S-phase entry by keeping p21 levels low, preventing premature S-phase exit upon DNA damage. Thus, we characterize how p21 regulates the proliferation-quiescence decision to maintain genomic stability. PMID:28317845

DNA damage and polyploidization.

PubMed

Chow, Jeremy; Poon, Randy Y C

2010-01-01

A growing body of evidence indicates that polyploidization triggers chromosomal instability and contributes to tumorigenesis. DNA damage is increasingly being recognized for its roles in promoting polyploidization. Although elegant mechanisms known as the DNA damage checkpoints are responsible for halting the cell cycle after DNA damage, agents that uncouple the checkpoints can induce unscheduled entry into mitosis. Likewise, defects of the checkpoints in several disorders permit mitotic entry even in the presence of DNA damage. Forcing cells with damaged DNA into mitosis causes severe chromosome segregation defects, including lagging chromosomes, chromosomal fragments and chromosomal bridges. The presence of these lesions in the cleavage plane is believed to abort cytokinesis. It is postulated that if cytokinesis failure is coupled with defects of the p53-dependent postmitotic checkpoint pathway, cells can enter S phase and become polyploids. Progress in the past several years has unraveled some of the underlying principles of these pathways and underscored the important role of DNA damage in polyploidization. Furthermore, polyploidization per se may also be an important determinant of sensitivity to DNA damage, thereby may offer an opportunity for novel therapies.

Phospho-ubiquitin: upending the PINK–Parkin–ubiquitin cascade

PubMed Central

Matsuda, Noriyuki

2016-01-01

Mitochondria with decreased membrane potential are characterized by defects in protein import into the matrix and impairments in high-efficiency synthesis of ATP. These low-quality mitochondria are marked with ubiquitin for selective degradation. Key factors in this mechanism are PTEN-induced putative kinase 1 (PINK1, a mitochondrial kinase) and Parkin (a ubiquitin ligase), disruption of which has been implicated in predisposition to Parkinson’s disease. Previously, the clearance of damaged mitochondria had been thought to be the end result of a simple cascading reaction of PINK1–Parkin–ubiquitin. However, in the past year, several research groups including ours unexpectedly revealed that Parkin regulation is mediated by PINK1-dependent phosphorylation of ubiquitin. These results overturned the simple hierarchy that posited PINK1 and ubiquitin as the upstream and downstream factors of Parkin, respectively. Although ubiquitylation is well-known as a post-translational modification, it has recently become clear that ubiquitin itself can be modified, and that this modification unexpectedly converts ubiquitin to a factor that functions in retrograde signalling. PMID:26839319

Strategic role of the ubiquitin-dependent segregase p97 (VCP or Cdc48) in DNA replication.

PubMed

Ramadan, Kristijan; Halder, Swagata; Wiseman, Katherine; Vaz, Bruno

2017-02-01

Genome amplification (DNA synthesis) is one of the most demanding cellular processes in all proliferative cells. The DNA replication machinery (also known as the replisome) orchestrates genome amplification during S-phase of the cell cycle. Genetic material is particularly vulnerable to various events that can challenge the replisome during its assembly, activation (firing), progression (elongation) and disassembly from chromatin (termination). Any disturbance of the replisome leads to stalling of the DNA replication fork and firing of dormant replication origins, a process known as DNA replication stress. DNA replication stress is considered to be one of the main causes of sporadic cancers and other pathologies related to tissue degeneration and ageing. The mechanisms of replisome assembly and elongation during DNA synthesis are well understood. However, once DNA synthesis is complete, the process of replisome disassembly, and its removal from chromatin, remains unclear. In recent years, a growing body of evidence has alluded to a central role in replisome regulation for the ubiquitin-dependent protein segregase p97, also known as valosin-containing protein (VCP) in metazoans and Cdc48 in lower eukaryotes. By orchestrating the spatiotemporal turnover of the replisome, p97 plays an essential role in DNA replication. In this review, we will summarise our current knowledge about how p97 controls the replisome from replication initiation, to elongation and finally termination. We will also further examine the more recent findings concerning the role of p97 and how mutations in p97 cofactors, also known as adaptors, cause DNA replication stress induced genomic instability that leads to cancer and accelerated ageing. To our knowledge, this is the first comprehensive review concerning the mechanisms involved in the regulation of DNA replication by p97.

The importance of regulatory ubiquitination in cancer and metastasis

PubMed Central

Gallo, L. H.; Ko, J.; Donoghue, D. J.

2017-01-01

ABSTRACT Ubiquitination serves as a degradation mechanism of proteins, but is involved in additional cellular processes such as activation of NFκB inflammatory response and DNA damage repair. We highlight the E2 ubiquitin conjugating enzymes, E3 ubiquitin ligases and Deubiquitinases that support the metastasis of a plethora of cancers. E3 ubiquitin ligases also modulate pluripotent cancer stem cells attributed to chemotherapy resistance. We further describe mutations in E3 ubiquitin ligases that support tumor proliferation and adaptation to hypoxia. Thus, this review describes how tumors exploit members of the vast ubiquitin signaling pathways to support aberrant oncogenic signaling for survival and metastasis. PMID:28166483

OTUB1 Co-opts Lys48-Linked Ubiquitin Recognition to Suppress E2 Enzyme Function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Juang, Yu-Chi; Landry, Marie-Claude; Sanches, Mario

2012-03-26

Ubiquitylation entails the concerted action of E1, E2, and E3 enzymes. We recently reported that OTUB1, a deubiquitylase, inhibits the DNA damage response independently of its isopeptidase activity. OTUB1 does so by blocking ubiquitin transfer by UBC13, the cognate E2 enzyme for RNF168. OTUB1 also inhibits E2s of the UBE2D and UBE2E families. Here we elucidate the structural mechanism by which OTUB1 binds E2s to inhibit ubiquitin transfer. OTUB1 recognizes ubiquitin-charged E2s through contacts with both donor ubiquitin and the E2 enzyme. Surprisingly, free ubiquitin associates with the canonical distal ubiquitin-binding site on OTUB1 to promote formation of the inhibitedmore » E2 complex. Lys48 of donor ubiquitin lies near the OTUB1 catalytic site and the C terminus of free ubiquitin, a configuration that mimics the products of Lys48-linked ubiquitin chain cleavage. OTUB1 therefore co-opts Lys48-linked ubiquitin chain recognition to suppress ubiquitin conjugation and the DNA damage response.« less

Phospho-ubiquitin: upending the PINK-Parkin-ubiquitin cascade.

PubMed

Matsuda, Noriyuki

2016-04-01

Mitochondria with decreased membrane potential are characterized by defects in protein import into the matrix and impairments in high-efficiency synthesis of ATP. These low-quality mitochondria are marked with ubiquitin for selective degradation. Key factors in this mechanism are PTEN-induced putative kinase 1 (PINK1, a mitochondrial kinase) and Parkin (a ubiquitin ligase), disruption of which has been implicated in predisposition to Parkinson's disease. Previously, the clearance of damaged mitochondria had been thought to be the end result of a simple cascading reaction of PINK1-Parkin-ubiquitin. However, in the past year, several research groups including ours unexpectedly revealed that Parkin regulation is mediated by PINK1-dependent phosphorylation of ubiquitin. These results overturned the simple hierarchy that posited PINK1 and ubiquitin as the upstream and downstream factors of Parkin, respectively. Although ubiquitylation is well-known as a post-translational modification, it has recently become clear that ubiquitin itself can be modified, and that this modification unexpectedly converts ubiquitin to a factor that functions in retrograde signalling. © The Authors 2016. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

The Ubiquitin Ligase CHIP Prevents SirT6 Degradation through Noncanonical Ubiquitination

PubMed Central

Ronnebaum, Sarah M.; Wu, Yaxu; McDonough, Holly

2013-01-01

The ubiquitin ligase CHIP (carboxyl terminus of Hsp70-interacting protein) regulates protein quality control, and CHIP deletion accelerates aging and reduces the life span in mice. Here, we reveal a mechanism for CHIP's influence on longevity by demonstrating that CHIP stabilizes the sirtuin family member SirT6, a lysine deacetylase/ADP ribosylase involved in DNA repair, metabolism, and longevity. In CHIP-deficient cells, SirT6 protein half-life is substantially reduced due to increased proteasome-mediated degradation, but CHIP overexpression in these cells increases SirT6 protein expression without affecting SirT6 transcription. CHIP noncanonically ubiquitinates SirT6 at K170, which stabilizes SirT6 and prevents SirT6 canonical ubiquitination by other ubiquitin ligases. In CHIP-depleted cells, SirT6 K170 mutation increases SirT6 half-life and prevents proteasome-mediated degradation. The global decrease in SirT6 expression in the absence of CHIP is associated with decreased SirT6 promoter occupancy, which increases histone acetylation and promotes downstream gene transcription in CHIP-depleted cells. Cells lacking CHIP are hypersensitive to DNA-damaging agents, but DNA repair and cell viability are rescued by enforced expression of SirT6. The discovery of this CHIP-SirT6 interaction represents a novel protein-stabilizing mechanism and defines an intersection between protein quality control and epigenetic regulation to influence pathways that regulate the biology of aging. PMID:24043303

HTLV-1 Tax Oncoprotein Subverts the Cellular DNA Damage Response via Binding to DNA-dependent Protein Kinase*S⃞

PubMed Central

Durkin, Sarah S.; Guo, Xin; Fryrear, Kimberly A.; Mihaylova, Valia T.; Gupta, Saurabh K.; Belgnaoui, S. Mehdi; Haoudi, Abdelali; Kupfer, Gary M.; Semmes, O. John

2008-01-01

Human T-cell leukemia virus type-1 is the causative agent for adult T-cell leukemia. Previous research has established that the viral oncoprotein Tax mediates the transformation process by impairing cell cycle control and cellular response to DNA damage. We showed previously that Tax sequesters huChk2 within chromatin and impairs the response to ionizing radiation. Here we demonstrate that DNA-dependent protein kinase (DNA-PK) is a member of the Tax·Chk2 nuclear complex. The catalytic subunit, DNA-PKcs, and the regulatory subunit, Ku70, were present. Tax-containing nuclear extracts showed increased DNA-PK activity, and specific inhibition of DNA-PK prevented Tax-induced activation of Chk2 kinase activity. Expression of Tax induced foci formation and phosphorylation of H2AX. However, Tax-induced constitutive signaling of the DNA-PK pathway impaired cellular response to new damage, as reflected in suppression of ionizing radiation-induced DNA-PK phosphorylation and γH2AX stabilization. Tax co-localized with phospho-DNA-PK into nuclear speckles and a nuclear excluded Tax mutant sequestered endogenous phospho-DNA-PK into the cytoplasm, suggesting that Tax interaction with DNA-PK is an initiating event. We also describe a novel interaction between DNA-PK and Chk2 that requires Tax. We propose that Tax binds to and stabilizes a protein complex with DNA-PK and Chk2, resulting in a saturation of DNA-PK-mediated damage repair response. PMID:18957425

TRAIP promotes DNA damage response during genome replication and is mutated in primordial dwarfism.

PubMed

Harley, Margaret E; Murina, Olga; Leitch, Andrea; Higgs, Martin R; Bicknell, Louise S; Yigit, Gökhan; Blackford, Andrew N; Zlatanou, Anastasia; Mackenzie, Karen J; Reddy, Kaalak; Halachev, Mihail; McGlasson, Sarah; Reijns, Martin A M; Fluteau, Adeline; Martin, Carol-Anne; Sabbioneda, Simone; Elcioglu, Nursel H; Altmüller, Janine; Thiele, Holger; Greenhalgh, Lynn; Chessa, Luciana; Maghnie, Mohamad; Salim, Mahmoud; Bober, Michael B; Nürnberg, Peter; Jackson, Stephen P; Hurles, Matthew E; Wollnik, Bernd; Stewart, Grant S; Jackson, Andrew P

2016-01-01

DNA lesions encountered by replicative polymerases threaten genome stability and cell cycle progression. Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism. We establish that TRAIP relocalizes to sites of DNA damage, where it is required for optimal phosphorylation of H2AX and RPA2 during S-phase in response to ultraviolet (UV) irradiation, as well as fork progression through UV-induced DNA lesions. TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes. Human genetics thus identifies TRAIP as a component of the DNA damage response to replication-blocking DNA lesions.

TRAIP promotes DNA damage response during genome replication and is mutated in primordial dwarfism

PubMed Central

Leitch, Andrea; Higgs, Martin R.; Bicknell, Louise S.; Yigit, Gökhan; Blackford, Andrew N.; Zlatanou, Anastasia; Mackenzie, Karen J.; Reddy, Kaalak; Halachev, Mihail; McGlasson, Sarah; Reijns, Martin A. M.; Fluteau, Adeline; Martin, Carol-Anne; Sabbioneda, Simone; Elcioglu, Nursel H.; Altmüller, Janine; Thiele, Holger; Greenhalgh, Lynn; Chessa, Luciana; Maghnie, Mohamad; Salim, Mahmoud; Bober, Michael B.; Nürnberg, Peter; Jackson, Stephen P.; Hurles, Matthew E.; Wollnik, Bernd; Stewart, Grant S.; Jackson, Andrew P.

2015-01-01

DNA lesions encountered by replicative polymerases threaten genome stability and cell cycle progression. Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism/Seckel syndrome. We establish that TRAIP relocalizes to sites of DNA damage where it is required for optimal phosphorylation of H2AX and RPA2 during S-phase in response to UV irradiation, as well as fork progression through UV-induced DNA lesions. TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes. Human genetics thus identifies TRAIP as a novel component of the DNA damage response to replication-blocking DNA lesions. PMID:26595769

WWOX, the common fragile site FRA16D gene product, regulates ATM activation and the DNA damage response

PubMed Central

Abu-Odeh, Mohammad; Salah, Zaidoun; Herbel, Christoph; Hofmann, Thomas G.; Aqeilan, Rami I.

2014-01-01

Genomic instability is a hallmark of cancer. The WW domain-containing oxidoreductase (WWOX) is a tumor suppressor spanning the common chromosomal fragile site FRA16D. Here, we report a direct role of WWOX in DNA damage response (DDR) and DNA repair. We show that Wwox deficiency results in reduced activation of the ataxia telangiectasia-mutated (ATM) checkpoint kinase, inefficient induction and maintenance of γ-H2AX foci, and impaired DNA repair. Mechanistically, we show that, upon DNA damage, WWOX accumulates in the cell nucleus, where it interacts with ATM and enhances its activation. Nuclear accumulation of WWOX is regulated by its K63-linked ubiquitination at lysine residue 274, which is mediated by the E3 ubiquitin ligase ITCH. These findings identify a novel role for the tumor suppressor WWOX and show that loss of WWOX expression may drive genomic instability and provide an advantage for clonal expansion of neoplastic cells. PMID:25331887

Ubiquitin-Dependent Degradation of Mitochondrial Proteins Regulates Energy Metabolism.

PubMed

Lavie, Julie; De Belvalet, Harmony; Sonon, Sessinou; Ion, Ana Madalina; Dumon, Elodie; Melser, Su; Lacombe, Didier; Dupuy, Jean-William; Lalou, Claude; Bénard, Giovanni

2018-06-05

The ubiquitin proteasome system (UPS) regulates many cellular functions by degrading key proteins. Notably, the role of UPS in regulating mitochondrial metabolic functions is unclear. Here, we show that ubiquitination occurs in different mitochondrial compartments, including the inner mitochondrial membrane, and that turnover of several metabolic proteins is UPS dependent. We specifically detailed mitochondrial ubiquitination and subsequent UPS-dependent degradation of succinate dehydrogenase subunit A (SDHA), which occurred when SDHA was minimally involved in mitochondrial energy metabolism. We demonstrate that SDHA ubiquitination occurs inside the organelle. In addition, we show that the specific inhibition of SDHA degradation by UPS promotes SDHA-dependent oxygen consumption and increases ATP, malate, and citrate levels. These findings suggest that the mitochondrial metabolic machinery is also regulated by the UPS. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

RPA-coated single-stranded DNA as a platform for post-translational modifications in the DNA damage response

PubMed Central

Maréchal, Alexandre; Zou, Lee

2015-01-01

The Replication Protein A (RPA) complex is an essential regulator of eukaryotic DNA metabolism. RPA avidly binds to single-stranded DNA (ssDNA) through multiple oligonucleotide/oligosaccharide-binding folds and coordinates the recruitment and exchange of genome maintenance factors to regulate DNA replication, recombination and repair. The RPA-ssDNA platform also constitutes a key physiological signal which activates the master ATR kinase to protect and repair stalled or collapsed replication forks during replication stress. In recent years, the RPA complex has emerged as a key target and an important regulator of post-translational modifications in response to DNA damage, which is critical for its genome guardian functions. Phosphorylation and SUMOylation of the RPA complex, and more recently RPA-regulated ubiquitination, have all been shown to control specific aspects of DNA damage signaling and repair by modulating the interactions between RPA and its partners. Here, we review our current understanding of the critical functions of the RPA-ssDNA platform in the maintenance of genome stability and its regulation through an elaborate network of covalent modifications. PMID:25403473

RPA-coated single-stranded DNA as a platform for post-translational modifications in the DNA damage response.

PubMed

Maréchal, Alexandre; Zou, Lee

2015-01-01

The Replication Protein A (RPA) complex is an essential regulator of eukaryotic DNA metabolism. RPA avidly binds to single-stranded DNA (ssDNA) through multiple oligonucleotide/oligosaccharide-binding folds and coordinates the recruitment and exchange of genome maintenance factors to regulate DNA replication, recombination and repair. The RPA-ssDNA platform also constitutes a key physiological signal which activates the master ATR kinase to protect and repair stalled or collapsed replication forks during replication stress. In recent years, the RPA complex has emerged as a key target and an important regulator of post-translational modifications in response to DNA damage, which is critical for its genome guardian functions. Phosphorylation and SUMOylation of the RPA complex, and more recently RPA-regulated ubiquitination, have all been shown to control specific aspects of DNA damage signaling and repair by modulating the interactions between RPA and its partners. Here, we review our current understanding of the critical functions of the RPA-ssDNA platform in the maintenance of genome stability and its regulation through an elaborate network of covalent modifications.

Functions of Ubiquitin and SUMO in DNA Replication and Replication Stress

PubMed Central

García-Rodríguez, Néstor; Wong, Ronald P.; Ulrich, Helle D.

2016-01-01

Complete and faithful duplication of its entire genetic material is one of the essential prerequisites for a proliferating cell to maintain genome stability. Yet, during replication DNA is particularly vulnerable to insults. On the one hand, lesions in replicating DNA frequently cause a stalling of the replication machinery, as most DNA polymerases cannot cope with defective templates. This situation is aggravated by the fact that strand separation in preparation for DNA synthesis prevents common repair mechanisms relying on strand complementarity, such as base and nucleotide excision repair, from working properly. On the other hand, the replication process itself subjects the DNA to a series of hazardous transformations, ranging from the exposure of single-stranded DNA to topological contortions and the generation of nicks and fragments, which all bear the risk of inducing genomic instability. Dealing with these problems requires rapid and flexible responses, for which posttranslational protein modifications that act independently of protein synthesis are particularly well suited. Hence, it is not surprising that members of the ubiquitin family, particularly ubiquitin itself and SUMO, feature prominently in controlling many of the defensive and restorative measures involved in the protection of DNA during replication. In this review we will discuss the contributions of ubiquitin and SUMO to genome maintenance specifically as they relate to DNA replication. We will consider cases where the modifiers act during regular, i.e., unperturbed stages of replication, such as initiation, fork progression, and termination, but also give an account of their functions in dealing with lesions, replication stalling and fork collapse. PMID:27242895

Molecular Basis for Impaired DNA Damage Response Function Associated with the RAP80 ΔE81 Defect*

PubMed Central

Anamika; Markin, Craig J.; Rout, Manoj K.; Spyracopoulos, Leo

2014-01-01

Signal transduction within the DNA damage response is driven by the flux of protein-protein interaction cascades that ultimately recruit repair complexes to sites of damage. The protein RAP80 plays a central role in the damage response by targeting BRCA1/BRCA2 tumor suppressors to DNA damage foci through multivalent binding of Lys-63-linked polyubiquitin chains. Mutations within the high penetrance BRCA1/BRCA2 genes account for ∼20% of familial breast cancers. The genetic basis for the remaining cancers remains unknown, but may involve defects in binding partners for BRCA1 and BRCA2 that lead to impaired targeting to foci and a concomitant role in the pathogenesis of cancer. Recently, an in-frame deletion mutation (ΔE81) in a conserved region from the first ubiquitin interaction motif of RAP80 has been linked to an increase in chromosomal abnormalities. Using NMR spectroscopy, we demonstrate that the N-cap motif within the α-helix of the first ubiquitin interaction motif from ΔE81 undergoes a structural frameshift that leads to abolishment of multivalent binding of polyubiquitin chains. Loss of this single glutamate residue disrupts favorable electrostatic interactions between RAP80 and ubiquitin, establishing a plausible molecular basis for a potential predisposition to cancer unrelated to mutations within BRCA1/BRCA2 genes. PMID:24627472

Posttranslational Regulation of Human DNA Polymerase ι.

PubMed

McIntyre, Justyna; McLenigan, Mary P; Frank, Ekaterina G; Dai, Xiaoxia; Yang, Wei; Wang, Yinsheng; Woodgate, Roger

2015-11-06

Human DNA polymerases (pols) η and ι are Y-family DNA polymerase paralogs that facilitate translesion synthesis past damaged DNA. Both polη and polι can be monoubiquitinated in vivo. Polη has been shown to be ubiquitinated at one primary site. When this site is unavailable, three nearby lysines may become ubiquitinated. In contrast, mass spectrometry analysis of monoubiquitinated polι revealed that it is ubiquitinated at over 27 unique sites. Many of these sites are localized in different functional domains of the protein, including the catalytic polymerase domain, the proliferating cell nuclear antigen-interacting region, the Rev1-interacting region, and its ubiquitin binding motifs UBM1 and UBM2. Polι monoubiquitination remains unchanged after cells are exposed to DNA-damaging agents such as UV light (generating UV photoproducts), ethyl methanesulfonate (generating alkylation damage), mitomycin C (generating interstrand cross-links), or potassium bromate (generating direct oxidative DNA damage). However, when exposed to naphthoquinones, such as menadione and plumbagin, which cause indirect oxidative damage through mitochondrial dysfunction, polι becomes transiently polyubiquitinated via Lys(11)- and Lys(48)-linked chains of ubiquitin and subsequently targeted for degradation. Polyubiquitination does not occur as a direct result of the perturbation of the redox cycle as no polyubiquitination was observed after treatment with rotenone or antimycin A, which both inhibit mitochondrial electron transport. Interestingly, polyubiquitination was observed after the inhibition of the lysine acetyltransferase KATB3/p300. We hypothesize that the formation of polyubiquitination chains attached to polι occurs via the interplay between lysine acetylation and ubiquitination of ubiquitin itself at Lys(11) and Lys(48) rather than oxidative damage per se. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Proteasome-dependent degradation of replisome components regulates faithful DNA replication.

PubMed

Roseaulin, Laura C; Noguchi, Chiaki; Noguchi, Eishi

2013-08-15

The replication machinery, or the replisome, collides with a variety of obstacles during the normal process of DNA replication. In addition to damaged template DNA, numerous chromosome regions are considered to be difficult to replicate owing to the presence of DNA secondary structures and DNA-binding proteins. Under these conditions, the replication fork stalls, generating replication stress. Stalled forks are prone to collapse, posing serious threats to genomic integrity. It is generally thought that the replication checkpoint functions to stabilize the replisome and replication fork structure upon replication stress. This is important in order to allow DNA replication to resume once the problem is solved. However, our recent studies demonstrated that some replisome components undergo proteasome-dependent degradation during DNA replication in the fission yeast Schizosaccharomyces pombe. Our investigation has revealed the involvement of the SCF(Pof3) (Skp1-Cullin/Cdc53-F-box) ubiquitin ligase in replisome regulation. We also demonstrated that forced accumulation of the replisome components leads to abnormal DNA replication upon replication stress. Here we review these findings and present additional data indicating the importance of replisome degradation for DNA replication. Our studies suggest that cells activate an alternative pathway to degrade replisome components in order to preserve genomic integrity.

Autophagy regulates DNA repair through SQSTM1/p62.

PubMed

Feng, Yuchen; Klionsky, Daniel J

2017-06-03

Macroautophagy/autophagy is primarily a degradative pathway that clears malfunctioning cellular components in response to various types of stress. Recent studies have indicated that autophagy also plays an important role in maintaining genome stability. Loss of autophagy is associated with increased damage to DNA, inappropriate amplification of genomic regions and abnormal chromosome number. In a recent paper by Wang et al. the authors uncover a mechanism through which autophagy regulates the ubiquitination of chromatin. In particular, the autophagy receptor and substrate SQSTM1/p62 inhibits the E3 ligase RNF168-dependent ubiquitination of histone in response to DNA double-strand breaks. Dysregulation of this process leads to a reduced ability to repair DNA and a corresponding increase in the sensitivity of cells to radiation-induced damage.

Interleukin-22 drives nitric oxide-dependent DNA damage and dysplasia in a murine model of colitis-associated cancer

PubMed Central

Sheh, A; Muthupalani, S; Bryant, EM; Puglisi, DA; Holcombe, H; Conaway, EA; Parry, NAP; Bakthavatchalu, V; Short, SP; Williams, CS; Wogan, GN; Tannenbaum, SR; Fox, JG; Horwitz, BH

2017-01-01

The risk of colon cancer is increased in patients with Crohn's disease and ulcerative colitis. Inflammation-induced DNA damage could be an important link between inflammation and cancer, although the pathways that link inflammation and DNA damage are incompletely defined. RAG2-deficient mice infected with Helicobacter hepaticus (Hh) develop colitis that progresses to lower bowel cancer. This process depends on nitric oxide (NO), a molecule with known mutagenic potential. We have previously hypothesized that production of NO by macrophages could be essential for Hh-driven carcinogenesis, however, whether Hh-infection induces DNA damage in this model and whether this depends on NO has not been determined. Here, we demonstrate that Hh infection of RAG2-deficient mice rapidly induces expression of iNOS and the development of DNA double-stranded breaks (DSBs) specifically in proliferating crypt epithelial cells. Generation of DSBs depended on iNOS activity, and further, induction of iNOS, the generation of DSBs, and the subsequent development of dysplasia were inhibited by depletion of the Hh-induced cytokine IL-22. These results demonstrate a strong association between Hh-induced DNA damage and the development of dysplasia, and further suggest that IL-22 dependent induction of iNOS within crypt epithelial cells rather than macrophages is a driving force in this process. PMID:28198364

RNF8 E3 Ubiquitin Ligase Stimulates Ubc13 E2 Conjugating Activity That Is Essential for DNA Double Strand Break Signaling and BRCA1 Tumor Suppressor Recruitment

DOE PAGES

Hodge, Curtis D.; Ismail, Ismail H.; Edwards, Ross A.; ...

2016-02-22

DNA double strand break (DSB) responses depend on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 plus E2 ubiquitin-conjugating enzyme Ubc13 to specifically generate histone Lys-63-linked ubiquitin chains in DSB signaling. In this paper, we defined the activated RNF8-Ubc13~ubiquitin complex by x-ray crystallography and its functional solution conformations by x-ray scattering, as tested by separation-of-function mutations imaged in cells by immunofluorescence. The collective results show that the RING E3 RNF8 targets E2 Ubc13 to DSB sites and plays a critical role in damage signaling by stimulating polyubiquitination through modulating conformations of ubiquitin covalently linked to the Ubc13more » active site. Structure-guided separation-of-function mutations show that the RNF8 E2 stimulating activity is essential for DSB signaling in mammalian cells and is necessary for downstream recruitment of 53BP1 and BRCA1. Chromatin-targeted RNF168 rescues 53BP1 recruitment involved in non-homologous end joining but not BRCA1 recruitment for homologous recombination. Finally, these findings suggest an allosteric approach to targeting the ubiquitin-docking cleft at the E2-E3 interface for possible interventions in cancer and chronic inflammation, and moreover, they establish an independent RNF8 role in BRCA1 recruitment.« less

Precision cancer therapy: profiting from tumor specific defects in the DNA damage tolerance system.

PubMed

Buoninfante, Olimpia Alessandra; Pilzecker, Bas; Aslam, Muhammad Assad; Zavrakidis, Ioannis; van der Wiel, Rianne; van de Ven, Marieke; van den Berk, Paul C M; Jacobs, Heinz

2018-04-10

DNA damage tolerance (DDT) enables replication to continue in the presence of a damaged template and constitutes a key step in DNA interstrand crosslink repair. In this way DDT minimizes replication stress inflicted by a wide range of endogenous and exogenous agents, and provides a critical first line defense against alkylating and platinating chemotherapeutics. Effective DDT strongly depends on damage-induced, site-specific PCNA-ubiquitination at Lysine (K) 164 by the E2/E3 complex (RAD6/18). A survey of The Cancer Genome Atlas (TCGA) revealed a high frequency of tumors presents RAD6/RAD18 bi-allelic inactivating deletions. For instance, 11% of renal cell carcinoma and 5% of pancreatic tumors have inactivating RAD18 -deletions and 7% of malignant peripheral nerve sheath tumors lack RAD6B . To determine the potential benefit for tumor-specific DDT defects, we followed a genetic approach by establishing unique sets of DDT-proficient Pcna K164 and -defective Pcna K164R lymphoma and breast cancer cell lines. In the absence of exogenous DNA damage, Pcna K164R tumors grew comparably to their Pcna K164 controls in vitro and in vivo . However, DDT-defective lymphomas and breast cancers were compared to their DDT-proficient controls hypersensitive to the chemotherapeutic drug cisplatin (CsPt), both in vitro and in vivo. CsPt strongly inhibited tumor growth and the overall survival of tumor bearing mice greatly improved in the DDT-defective condition. These insights open new therapeutic possibilities for precision cancer medicine with DNA damaging chemotherapeutics and optimize Next-Generation-Sequencing (NGS)-based cancer-diagnostics, -therapeutics, and -prognosis.

Precision cancer therapy: profiting from tumor specific defects in the DNA damage tolerance system

PubMed Central

Buoninfante, Olimpia Alessandra; Pilzecker, Bas; Aslam, Muhammad Assad; Zavrakidis, Ioannis; van der Wiel, Rianne; van de Ven, Marieke; van den Berk, Paul C.M.; Jacobs, Heinz

2018-01-01

DNA damage tolerance (DDT) enables replication to continue in the presence of a damaged template and constitutes a key step in DNA interstrand crosslink repair. In this way DDT minimizes replication stress inflicted by a wide range of endogenous and exogenous agents, and provides a critical first line defense against alkylating and platinating chemotherapeutics. Effective DDT strongly depends on damage-induced, site-specific PCNA-ubiquitination at Lysine (K) 164 by the E2/E3 complex (RAD6/18). A survey of The Cancer Genome Atlas (TCGA) revealed a high frequency of tumors presents RAD6/RAD18 bi-allelic inactivating deletions. For instance, 11% of renal cell carcinoma and 5% of pancreatic tumors have inactivating RAD18-deletions and 7% of malignant peripheral nerve sheath tumors lack RAD6B. To determine the potential benefit for tumor-specific DDT defects, we followed a genetic approach by establishing unique sets of DDT-proficient PcnaK164 and -defective PcnaK164R lymphoma and breast cancer cell lines. In the absence of exogenous DNA damage, PcnaK164R tumors grew comparably to their PcnaK164 controls in vitro and in vivo. However, DDT-defective lymphomas and breast cancers were compared to their DDT-proficient controls hypersensitive to the chemotherapeutic drug cisplatin (CsPt), both in vitro and in vivo. CsPt strongly inhibited tumor growth and the overall survival of tumor bearing mice greatly improved in the DDT-defective condition. These insights open new therapeutic possibilities for precision cancer medicine with DNA damaging chemotherapeutics and optimize Next-Generation-Sequencing (NGS)-based cancer-diagnostics, -therapeutics, and -prognosis. PMID:29721165

Pulsewidth-dependent nature of laser-induced DNA damage in RPE cells

NASA Astrophysics Data System (ADS)

Hall, Rebecca M.; Glickman, Randolph D.; Rockwell, Benjamin A.; Kumar, Neeru; Noojin, Gary D.

2001-07-01

Ultrashort pulse laser radiation may produce cellular damage through unique mechanisms. Primary cultures of bovine retinal pigment epithelial (RPE) cells were exposed to the out put of a Ti:Sapphire laser producing 30 fs (mode-locked) pulses, 44 amplified fs pulses, or continuous wave exposures at 800 nm. Laser exposures at and below the damage threshold were studied. DNA damage was detected using single cell gel electrophoresis (comet assay). Unexposed (control) cells produced short tails with low tail moments. In contrast, all laser-exposed cells showed some degree of DNA fragmentation, but the size and shape of the resulting comets differed among the various modalities. CW-exposed cells produced generally light and relatively compact tails, suggesting fewer and larger DNA fragments, while mode-locked laser exposures (30 fs pulses) resulted in large and diffuse comets, indicating the DNA was fragmented into many very small pieces. Work is continuing to define the relationship of laser pulsewidth and intensity with the degree of DNA fragmentation. These results suggest that DNA damage may result from multiple mechanisms of laser-cell interaction, including multiphoton absorption.

Regulation of ATM-Dependent DNA Damage Responses in Breast Cancer by the RhoGEF Net1

DTIC Science & Technology

2013-04-01

Science 279: 509-514. 5. Jaffe AB. et al., (2010) RhoGTPases: Biochemistry and Biology. Annu. Rev. Cell Dev. Biol. 21:247-269. 6. Rossman KL, et al...exchange factor Net1 is regulated by nuclear sequestration. J. Biol. Chem. 277:17, 14581-14588. 17. Harper JW, et al., (2007) The DNA Damage Response: Ten...Research (AACR) Annual Meeting and 2013 Annual Cancer Research Biochemistry Retreat Regulation of ATM-dependent DNA damage signaling in human breast

The ubiquitin family meets the Fanconi anemia proteins.

PubMed

Renaudin, Xavier; Koch Lerner, Leticia; Menck, Carlos Frederico Martins; Rosselli, Filippo

2016-01-01

Fanconi anaemia (FA) is a hereditary disorder characterized by bone marrow failure, developmental defects, predisposition to cancer and chromosomal abnormalities. FA is caused by biallelic mutations that inactivate genes encoding proteins involved in replication stress-associated DNA damage responses. The 20 FANC proteins identified to date constitute the FANC pathway. A key event in this pathway involves the monoubiquitination of the FANCD2-FANCI heterodimer by the collective action of at least 10 different proteins assembled in the FANC core complex. The FANC core complex-mediated monoubiquitination of FANCD2-FANCI is essential to assemble the heterodimer in subnuclear, chromatin-associated, foci and to regulate the process of DNA repair as well as the rescue of stalled replication forks. Several recent works have demonstrated that the activity of the FANC pathway is linked to several other protein post-translational modifications from the ubiquitin-like family, including SUMO and NEDD8. These modifications are related to DNA damage responses but may also affect other cellular functions potentially related to the clinical phenotypes of the syndrome. This review summarizes the interplay between the ubiquitin and ubiquitin-like proteins and the FANC proteins that constitute a major pathway for the surveillance of the genomic integrity and addresses the implications of their interactions in maintaining genome stability. Copyright © 2016 Elsevier B.V. All rights reserved.

Recruitment of TRF2 to laser-induced DNA damage sites.

PubMed

Huda, Nazmul; Abe, Satoshi; Gu, Ling; Mendonca, Marc S; Mohanty, Samarendra; Gilley, David

2012-09-01

Several lines of evidence suggest that the telomere-associated protein TRF2 plays critical roles in the DNA damage response. TRF2 is rapidly and transiently phosphorylated by an ATM-dependent pathway in response to DNA damage and this DNA damage-induced phosphoryation is essential for the DNA-PK-dependent pathway of DNA double-strand break repair (DSB). However, the type of DNA damage that induces TRF2 localization to the damage sites, the requirement for DNA damage-induced phosphorylation of TRF2 for its recruitment, as well as the detailed kinetics of TRF2 accumulation at DNA damage sites have not been fully investigated. In order to address these questions, we used an ultrafast femtosecond multiphoton laser and a continuous wave 405-nm single photon laser to induce DNA damage at defined nuclear locations. Our results showed that DNA damage produced by a femtosecond multiphoton laser was sufficient for localization of TRF2 to these DNA damage sites. We also demonstrate that ectopically expressed TRF2 was recruited to DNA lesions created by a 405-nm laser. Our data suggest that ATM and DNA-PKcs kinases are not required for TRF2 localization to DNA damage sites. Furthermore, we found that phosphorylation of TRF2 at residue T188 was not essential for its recruitment to laser-induced DNA damage sites. Thus, we provide further evidence that a protein known to function in telomere maintenance, TRF2, is recruited to sites of DNA damage and plays critical roles in the DNA damage response. Copyright © 2012 Elsevier Inc. All rights reserved.

Cell cycle stage-specific roles of Rad18 in tolerance and repair of oxidative DNA damage

PubMed Central

Yang, Yang; Durando, Michael; Smith-Roe, Stephanie L.; Sproul, Chris; Greenwalt, Alicia M.; Kaufmann, William; Oh, Sehyun; Hendrickson, Eric A.; Vaziri, Cyrus

2013-01-01

The E3 ubiquitin ligase Rad18 mediates tolerance of replication fork-stalling bulky DNA lesions, but whether Rad18 mediates tolerance of bulky DNA lesions acquired outside S-phase is unclear. Using synchronized cultures of primary human cells, we defined cell cycle stage-specific contributions of Rad18 to genome maintenance in response to ultraviolet C (UVC) and H2O2-induced DNA damage. UVC and H2O2 treatments both induced Rad18-mediated proliferating cell nuclear antigen mono-ubiquitination during G0, G1 and S-phase. Rad18 was important for repressing H2O2-induced (but not ultraviolet-induced) double strand break (DSB) accumulation and ATM S1981 phosphorylation only during G1, indicating a specific role for Rad18 in processing of oxidative DNA lesions outside S-phase. However, H2O2-induced DSB formation in Rad18-depleted G1 cells was not associated with increased genotoxin sensitivity, indicating that back-up DSB repair mechanisms compensate for Rad18 deficiency. Indeed, in DNA LigIV-deficient cells Rad18-depletion conferred H2O2-sensitivity, demonstrating functional redundancy between Rad18 and non-homologous end joining for tolerance of oxidative DNA damage acquired during G1. In contrast with G1-synchronized cultures, S-phase cells were H2O2-sensitive following Rad18-depletion. We conclude that although Rad18 pathway activation by oxidative lesions is not restricted to S-phase, Rad18-mediated trans-lesion synthesis by Polη is dispensable for damage-tolerance in G1 (because of back-up non-homologous end joining-mediated DSB repair), yet Rad18 is necessary for damage tolerance during S-phase. PMID:23295675

The Fanconi Anemia DNA Repair Pathway Is Regulated by an Interaction between Ubiquitin and the E2-like Fold Domain of FANCL*

PubMed Central

Miles, Jennifer A.; Frost, Mark G.; Carroll, Eilis; Rowe, Michelle L.; Howard, Mark J.; Sidhu, Ateesh; Chaugule, Viduth K.; Alpi, Arno F.; Walden, Helen

2015-01-01

The Fanconi Anemia (FA) DNA repair pathway is essential for the recognition and repair of DNA interstrand crosslinks (ICL). Inefficient repair of these ICL can lead to leukemia and bone marrow failure. A critical step in the pathway is the monoubiquitination of FANCD2 by the RING E3 ligase FANCL. FANCL comprises 3 domains, a RING domain that interacts with E2 conjugating enzymes, a central domain required for substrate interaction, and an N-terminal E2-like fold (ELF) domain. The ELF domain is found in all FANCL homologues, yet the function of the domain remains unknown. We report here that the ELF domain of FANCL is required to mediate a non-covalent interaction between FANCL and ubiquitin. The interaction involves the canonical Ile44 patch on ubiquitin, and a functionally conserved patch on FANCL. We show that the interaction is not necessary for the recognition of the core complex, it does not enhance the interaction between FANCL and Ube2T, and is not required for FANCD2 monoubiquitination in vitro. However, we demonstrate that the ELF domain is required to promote efficient DNA damage-induced FANCD2 monoubiquitination in vertebrate cells, suggesting an important function of ubiquitin binding by FANCL in vivo. PMID:26149689

Functional assessment of ubiquitin-depended processes under microgravity conditions

NASA Astrophysics Data System (ADS)

Zhabereva, Anastasia; Shenkman, Boris S.; Gainullin, Murat; Gurev, Eugeny; Kondratieva, Ekaterina; Kopylov, Arthur

Ubiquitylation, a widespread and important posttranslational modification of eukaryotic proteins, controls a multitude of critical cellular processes, both in normal and pathological conditions. The present work aims to study involvement of ubiquitin-dependent regulation in adaptive response to the external stimuli. Experiments were carried out on C57BL/6 mice. The microgravity state under conditions of real spaceflight on the biosatellite “BION-M1” was used as a model of stress impact. Additionally, number of control series including the vivarium control and experiments in Ground-based analog were also studied. The aggregate of endogenously ubiquitylated proteins was selected as specific feature of ubiquitin-dependent processes. Dynamic changes of modification pattern were characterized in liver tissue by combination of some methods, particularly by specific isolation of explicit protein pool, followed by immunodetection and/or mass spectrometry-based identification. The main approach includes specific extraction of proteins, modified by multiubiquitin chains of different length and topology. For this purpose two techniques were applied: 1) immunoprecipitation with antibodies against ubiquitin and/or multiubiquitin chains; 2) pull-down using synthetic protein construct termed Tandem Ubiquitin Binding Entities (TUBE, LifeSensors). TUBE represents fusion protein, composed of well characterized ubiquitin-binding domains, and thereby allows specific high-affinity binding and extraction of ubiquitylated proteins. Resulting protein fractions were analyzed by immunoblotting with antibodies against different types of multiubiquitin chains. Using this method we mapped endogenously modified proteins involved in two different types of ubiquitin-dependent processes, namely catabolic and non-catabolic ubiquitylation, in liver tissues, obtained from both control as well as experimental groups of animals, mentioned above. Then, isolated fractions of ubiquitylated proteins

Mule determines the apoptotic response to HDAC inhibitors by targeted ubiquitination and destruction of HDAC2

PubMed Central

Zhang, Jing; Kan, Shu; Huang, Brian; Hao, Zhenyue; Mak, Tak W.; Zhong, Qing

2011-01-01

Histone deacetylases (HDACs) are major epigenetic modulators involved in a broad spectrum of human diseases including cancers. Administration of HDAC inhibitors (HDACis) leads to growth inhibition, differentiation, and apoptosis of cancer cells. Understanding the regulatory mechanism of HDACs is imperative to harness the therapeutic potentials of HDACis. Here we show that HDACi- and DNA damage-induced apoptosis are severely compromised in mouse embryonic fibroblasts lacking a HECT domain ubiquitin ligase, Mule (Mcl-1 ubiquitin ligase E3). Mule specifically targets HDAC2 for ubiquitination and degradation. Accumulation of HDAC2 in Mule-deficient cells leads to compromised p53 acetylation as well as crippled p53 transcriptional activation, accumulation, and apoptotic response upon DNA damage and Nutlin-3 treatments. These defects in Mule-null cells can be partially reversed by HDACis and fully rescued by lowering the elevated HDAC2 in Mule-null cells to the normal levels as in wild-type cells. Taken together, our results reveal a critical regulatory mechanism of HDAC2 by Mule and suggest this pathway determines the cellular response to HDACis and DNA damage. PMID:22016339

Chromosome territories reposition during DNA damage-repair response

PubMed Central

2013-01-01

Background Local higher-order chromatin structure, dynamics and composition of the DNA are known to determine double-strand break frequencies and the efficiency of repair. However, how DNA damage response affects the spatial organization of chromosome territories is still unexplored. Results Our report investigates the effect of DNA damage on the spatial organization of chromosome territories within interphase nuclei of human cells. We show that DNA damage induces a large-scale spatial repositioning of chromosome territories that are relatively gene dense. This response is dose dependent, and involves territories moving from the nuclear interior to the periphery and vice versa. Furthermore, we have found that chromosome territory repositioning is contingent upon double-strand break recognition and damage sensing. Importantly, our results suggest that this is a reversible process where, following repair, chromosome territories re-occupy positions similar to those in undamaged control cells. Conclusions Thus, our report for the first time highlights DNA damage-dependent spatial reorganization of whole chromosomes, which might be an integral aspect of cellular damage response. PMID:24330859

An Interaction Landscape of Ubiquitin Signaling.

PubMed

Zhang, Xiaofei; Smits, Arne H; van Tilburg, Gabrielle B A; Jansen, Pascal W T C; Makowski, Matthew M; Ovaa, Huib; Vermeulen, Michiel

2017-03-02

Intracellular signaling via the covalent attachment of different ubiquitin linkages to protein substrates is fundamental to many cellular processes. Although linkage-selective ubiquitin interactors have been studied on a case-by-case basis, proteome-wide analyses have not been conducted yet. Here, we present ubiquitin interactor affinity enrichment-mass spectrometry (UbIA-MS), a quantitative interaction proteomics method that makes use of chemically synthesized diubiquitin to enrich and identify ubiquitin linkage interactors from crude cell lysates. UbIA-MS reveals linkage-selective diubiquitin interactions in multiple cell types. For example, we identify TAB2 and TAB3 as novel K6 diubiquitin interactors and characterize UCHL3 as a K27-linkage selective interactor that regulates K27 polyubiquitin chain formation in cells. Additionally, we show a class of monoubiquitin and K6 diubiquitin interactors whose binding is induced by DNA damage. We expect that our proteome-wide diubiquitin interaction landscape and established workflows will have broad applications in the ongoing efforts to decipher the complex language of ubiquitin signaling. Copyright © 2017 Elsevier Inc. All rights reserved.

DNA repair factor XPC is modified by SUMO-1 and ubiquitin following UV irradiation

PubMed Central

Wang, Qi-En; Zhu, Qianzheng; Wani, Gulzar; El-Mahdy, Mohamed A.; Li, Jinyou; Wani, Altaf A.

2005-01-01

Nucleotide excision repair (NER) is the major DNA repair process that removes diverse DNA lesions including UV-induced photoproducts. There are more than 20 proteins involved in NER. Among them, XPC is thought to be one of the first proteins to recognize DNA damage during global genomic repair (GGR), a sub-pathway of NER. In order to study the mechanism through which XPC participates in GGR, we investigated the possible modifications of XPC protein upon UV irradiation in mammalian cells. Western blot analysis of cell lysates from UV-irradiated normal human fibroblast, prepared by direct boiling in an SDS lysis buffer, showed several anti-XPC antibody-reactive bands with molecular weight higher than the original XPC protein. The reciprocal immunoprecipitation and siRNA transfection analysis demonstrated that XPC protein is modified by SUMO-1 and ubiquitin. By using several NER-deficient cell lines, we found that DDB2 and XPA are required for UV-induced XPC modifications. Interestingly, both the inactivation of ubiquitylation and the treatment of proteasome inhibitors quantitatively inhibited the UV-induced XPC modifications. Furthermore, XPC protein is degraded significantly following UV irradiation in XP-A cells in which sumoylation of XPC does not occur. Taken together, we conclude that XPC protein is modified by SUMO-1 and ubiquitin following UV irradiation and these modifications require the functions of DDB2 and XPA, as well as the ubiquitin–proteasome system. Our results also suggest that at least one function of UV-induced XPC sumoylation is related to the stabilization of XPC protein. PMID:16030353

Specificity and disease in the ubiquitin system

PubMed Central

Chaugule, Viduth K.; Walden, Helen

2016-01-01

Post-translational modification (PTM) of proteins by ubiquitination is an essential cellular regulatory process. Such regulation drives the cell cycle and cell division, signalling and secretory pathways, DNA replication and repair processes and protein quality control and degradation pathways. A huge range of ubiquitin signals can be generated depending on the specificity and catalytic activity of the enzymes required for attachment of ubiquitin to a given target. As a consequence of its importance to eukaryotic life, dysfunction in the ubiquitin system leads to many disease states, including cancers and neurodegeneration. This review takes a retrospective look at our progress in understanding the molecular mechanisms that govern the specificity of ubiquitin conjugation. PMID:26862208

The plant homeodomain fingers of fission yeast Msc1 exhibit E3 ubiquitin ligase activity.

PubMed

Dul, Barbara E; Walworth, Nancy C

2007-06-22

The DNA damage checkpoint pathway governs how cells regulate cell cycle progression in response to DNA damage. A screen for suppressors of a fission yeast chk1 mutant defective in the checkpoint pathway identified a novel Schizosaccharomyces pombe protein, Msc1. Msc1 contains 3 plant homeodomain (PHD) finger motifs, characteristically defined by a C4HC3 consensus similar to RING finger domains. PHD finger domains in viral proteins and in the cellular protein kinase MEKK1 (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1) have been implicated as ubiquitin E3 protein ligases that affect protein stability. The close structural relationship of PHD fingers to RING fingers suggests that other PHD domain-containing proteins might share this activity. We show that each of the three PHD fingers of Msc1 can act as ubiquitin E3 ligases, reporting for the first time that PHD fingers from a nuclear protein exhibit E3 ubiquitin ligase activity. The function of the PHD fingers of Msc1 is needed to rescue the DNA damage sensitivity of a chk1Delta strain. Msc1 co-precipitates Rhp6, the S. pombe homologue of the human ubiquitin-conjugating enzyme Ubc2. Strikingly, deletion of msc1 confers complete suppression of the slow growth phenotype, UV and hydroxyurea sensitivities of an rhp6 deletion strain and restores deficient histone H3 methylation observed in the rhp6Delta mutant. We speculate that the target of the E3 ubiquitin ligase activity of Msc1 is likely to be a chromatin-associated protein.

Ubiquitin mediates the physical and functional interaction between human DNA polymerases η and ι

PubMed Central

McIntyre, Justyna; Vidal, Antonio E.; McLenigan, Mary P.; Bomar, Martha G.; Curti, Elena; McDonald, John P.; Plosky, Brian S.; Ohashi, Eiji; Woodgate, Roger

2013-01-01

Human DNA polymerases η and ι are best characterized for their ability to facilitate translesion DNA synthesis (TLS). Both polymerases (pols) co-localize in ‘replication factories’ in vivo after cells are exposed to ultraviolet light and this co-localization is mediated through a physical interaction between the two TLS pols. We have mapped the polη-ι interacting region to their respective ubiquitin-binding domains (UBZ in polη and UBM1 and UBM2 in polι), and demonstrate that ubiquitination of either TLS polymerase is a prerequisite for their physical and functional interaction. Importantly, while monoubiquitination of polη precludes its ability to interact with proliferating cell nuclear antigen (PCNA), it enhances its interaction with polι. Furthermore, a polι-ubiquitin chimera interacts avidly with both polη and PCNA. Thus, the ubiquitination status of polη, or polι plays a key regulatory function in controlling the protein partners with which each polymerase interacts, and in doing so, determines the efficiency of targeting the respective polymerase to stalled replication forks where they facilitate TLS. PMID:23248005

FAAP20: a novel ubiquitin-binding FA nuclear core-complex protein required for functional integrity of the FA-BRCA DNA repair pathway

PubMed Central

Ali, Abdullah Mahmood; Pradhan, Arun; Singh, Thiyam Ramsingh; Du, Changhu; Li, Jie; Wahengbam, Kebola; Grassman, Elke; Auerbach, Arleen D.; Pang, Qishen

2012-01-01

Fanconi anemia (FA) nuclear core complex is a multiprotein complex required for the functional integrity of the FA-BRCA pathway regulating DNA repair. This pathway is inactivated in FA, a devastating genetic disease, which leads to hematologic defects and cancer in patients. Here we report the isolation and characterization of a novel 20-kDa FANCA-associated protein (FAAP20). We show that FAAP20 is an integral component of the FA nuclear core complex. We identify a region on FANCA that physically interacts with FAAP20, and show that FANCA regulates stability of this protein. FAAP20 contains a conserved ubiquitin-binding zinc-finger domain (UBZ), and binds K-63–linked ubiquitin chains in vitro. The FAAP20-UBZ domain is not required for interaction with FANCA, but is required for DNA-damage–induced chromatin loading of FANCA and the functional integrity of the FA pathway. These findings reveal critical roles for FAAP20 in the FA-BRCA pathway of DNA damage repair and genome maintenance. PMID:22343915

The Fanconi Anemia DNA Repair Pathway Is Regulated by an Interaction between Ubiquitin and the E2-like Fold Domain of FANCL.

PubMed

Miles, Jennifer A; Frost, Mark G; Carroll, Eilis; Rowe, Michelle L; Howard, Mark J; Sidhu, Ateesh; Chaugule, Viduth K; Alpi, Arno F; Walden, Helen

2015-08-21

The Fanconi Anemia (FA) DNA repair pathway is essential for the recognition and repair of DNA interstrand crosslinks (ICL). Inefficient repair of these ICL can lead to leukemia and bone marrow failure. A critical step in the pathway is the monoubiquitination of FANCD2 by the RING E3 ligase FANCL. FANCL comprises 3 domains, a RING domain that interacts with E2 conjugating enzymes, a central domain required for substrate interaction, and an N-terminal E2-like fold (ELF) domain. The ELF domain is found in all FANCL homologues, yet the function of the domain remains unknown. We report here that the ELF domain of FANCL is required to mediate a non-covalent interaction between FANCL and ubiquitin. The interaction involves the canonical Ile44 patch on ubiquitin, and a functionally conserved patch on FANCL. We show that the interaction is not necessary for the recognition of the core complex, it does not enhance the interaction between FANCL and Ube2T, and is not required for FANCD2 monoubiquitination in vitro. However, we demonstrate that the ELF domain is required to promote efficient DNA damage-induced FANCD2 monoubiquitination in vertebrate cells, suggesting an important function of ubiquitin binding by FANCL in vivo. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Rad18 confers hematopoietic progenitor cell DNA damage tolerance independently of the Fanconi Anemia pathway in vivo

PubMed Central

Yang, Yang; Poe, Jonathan C.; Yang, Lisong; Fedoriw, Andrew; Desai, Siddhi; Magnuson, Terry; Li, Zhiguo; Fedoriw, Yuri; Araki, Kimi; Gao, Yanzhe; Tateishi, Satoshi; Sarantopoulos, Stefanie; Vaziri, Cyrus

2016-01-01

In cultured cancer cells the E3 ubiquitin ligase Rad18 activates Trans-Lesion Synthesis (TLS) and the Fanconi Anemia (FA) pathway. However, physiological roles of Rad18 in DNA damage tolerance and carcinogenesis are unknown and were investigated here. Primary hematopoietic stem and progenitor cells (HSPC) co-expressed RAD18 and FANCD2 proteins, potentially consistent with a role for Rad18 in FA pathway function during hematopoiesis. However, hematopoietic defects typically associated with fanc-deficiency (decreased HSPC numbers, reduced engraftment potential of HSPC, and Mitomycin C (MMC) -sensitive hematopoiesis), were absent in Rad18−/− mice. Moreover, primary Rad18−/− mouse embryonic fibroblasts (MEF) retained robust Fancd2 mono-ubiquitination following MMC treatment. Therefore, Rad18 is dispensable for FA pathway activation in untransformed cells and the Rad18 and FA pathways are separable in hematopoietic cells. In contrast with responses to crosslinking agents, Rad18−/− HSPC were sensitive to in vivo treatment with the myelosuppressive agent 7,12 Dimethylbenz[a]anthracene (DMBA). Rad18-deficient fibroblasts aberrantly accumulated DNA damage markers after DMBA treatment. Moreover, in vivo DMBA treatment led to increased incidence of B cell malignancy in Rad18−/− mice. These results identify novel hematopoietic functions for Rad18 and provide the first demonstration that Rad18 confers DNA damage tolerance and tumor-suppression in a physiological setting. PMID:26883629

Mule determines the apoptotic response to HDAC inhibitors by targeted ubiquitination and destruction of HDAC2.

PubMed

Zhang, Jing; Kan, Shu; Huang, Brian; Hao, Zhenyue; Mak, Tak W; Zhong, Qing

2011-12-15

Histone deacetylases (HDACs) are major epigenetic modulators involved in a broad spectrum of human diseases including cancers. Administration of HDAC inhibitors (HDACis) leads to growth inhibition, differentiation, and apoptosis of cancer cells. Understanding the regulatory mechanism of HDACs is imperative to harness the therapeutic potentials of HDACis. Here we show that HDACi- and DNA damage-induced apoptosis are severely compromised in mouse embryonic fibroblasts lacking a HECT domain ubiquitin ligase, Mule (Mcl-1 ubiquitin ligase E3). Mule specifically targets HDAC2 for ubiquitination and degradation. Accumulation of HDAC2 in Mule-deficient cells leads to compromised p53 acetylation as well as crippled p53 transcriptional activation, accumulation, and apoptotic response upon DNA damage and Nutlin-3 treatments. These defects in Mule-null cells can be partially reversed by HDACis and fully rescued by lowering the elevated HDAC2 in Mule-null cells to the normal levels as in wild-type cells. Taken together, our results reveal a critical regulatory mechanism of HDAC2 by Mule and suggest this pathway determines the cellular response to HDACis and DNA damage. © 2011 by Cold Spring Harbor Laboratory Press

Squalene Inhibits ATM-Dependent Signaling in γIR-Induced DNA Damage Response through Induction of Wip1 Phosphatase.

PubMed

Tatewaki, Naoto; Konishi, Tetsuya; Nakajima, Yuki; Nishida, Miyako; Saito, Masafumi; Eitsuka, Takahiro; Sakamaki, Toshiyuki; Ikekawa, Nobuo; Nishida, Hiroshi

2016-01-01

Ataxia telangiectasia mutated (ATM) kinase plays a crucial role as a master controller in the cellular DNA damage response. Inhibition of ATM leads to inhibition of the checkpoint signaling pathway. Hence, addition of checkpoint inhibitors to anticancer therapies may be an effective targeting strategy. A recent study reported that Wip1, a protein phosphatase, de-phosphorylates serine 1981 of ATM during the DNA damage response. Squalene has been proposed to complement anticancer therapies such as chemotherapy and radiotherapy; however, there is little mechanistic information supporting this idea. Here, we report the inhibitory effect of squalene on ATM-dependent DNA damage signals. Squalene itself did not affect cell viability and the cell cycle of A549 cells, but it enhanced the cytotoxicity of gamma-irradiation (γIR). The in vitro kinase activity of ATM was not altered by squalene. However, squalene increased Wip1 expression in cells and suppressed ATM activation in γIR-treated cells. Consistent with the potential inhibition of ATM by squalene, IR-induced phosphorylation of ATM effectors such as p53 (Ser15) and Chk1 (Ser317) was inhibited by cell treatment with squalene. Thus, squalene inhibits the ATM-dependent signaling pathway following DNA damage through intracellular induction of Wip1 expression.

Selective autophagy: ubiquitin-mediated recognition and beyond.

PubMed

Kraft, Claudine; Peter, Matthias; Hofmann, Kay

2010-09-01

Eukaryotic cells use autophagy and the ubiquitin-proteasome system as their major protein degradation pathways. Whereas the ubiquitin-proteasome system is involved in the rapid degradation of proteins, autophagy pathways can selectively remove protein aggregates and damaged or excess organelles. Proteasome-mediated degradation requires previous ubiquitylation of the cargo, which is then recognized by ubiquitin receptors directing it to 26S proteasomes. Although autophagy has long been viewed as a random cytoplasmic degradation system, the involvement of ubiquitin as a specificity factor for selective autophagy is rapidly emerging. Recent evidence also suggests active crosstalk between proteasome-mediated degradation and selective autophagy. Here, we discuss the molecular mechanisms that link autophagy and the proteasome system, as well as the emerging roles of ubiquitin and ubiquitin-binding proteins in selective autophagy. On the basis of the evolutionary history of autophagic ubiquitin receptors, we propose a common origin for metazoan ubiquitin-dependent autophagy and the cytoplasm-to-vacuole targeting pathway of yeast.

Orchestration of DNA Damage Checkpoint Dynamics across the Human Cell Cycle.

PubMed

Chao, Hui Xiao; Poovey, Cere E; Privette, Ashley A; Grant, Gavin D; Chao, Hui Yan; Cook, Jeanette G; Purvis, Jeremy E

2017-11-22

Although molecular mechanisms that prompt cell-cycle arrest in response to DNA damage have been elucidated, the systems-level properties of DNA damage checkpoints are not understood. Here, using time-lapse microscopy and simulations that model the cell cycle as a series of Poisson processes, we characterize DNA damage checkpoints in individual, asynchronously proliferating cells. We demonstrate that, within early G1 and G2, checkpoints are stringent: DNA damage triggers an abrupt, all-or-none cell-cycle arrest. The duration of this arrest correlates with the severity of DNA damage. After the cell passes commitment points within G1 and G2, checkpoint stringency is relaxed. By contrast, all of S phase is comparatively insensitive to DNA damage. This checkpoint is graded: instead of halting the cell cycle, increasing DNA damage leads to slower S phase progression. In sum, we show that a cell's response to DNA damage depends on its exact cell-cycle position and that checkpoints are phase-dependent, stringent or relaxed, and graded or all-or-none. Copyright © 2017 Elsevier Inc. All rights reserved.

ATM-dependent phosphorylation of Mdm2 on serine 395: role in p53 activation by DNA damage

PubMed Central

Maya, Ruth; Balass, Moshe; Kim, Seong-Tae; Shkedy, Dganit; Leal, Juan-Fernando Martinez; Shifman, Ohad; Moas, Miri; Buschmann, Thomas; Ronai, Ze'ev; Shiloh, Yosef; Kastan, Michael B.; Katzir, Ephraim; Oren, Moshe

2001-01-01

The p53 tumor suppressor protein, a key regulator of cellular responses to genotoxic stress, is stabilized and activated after DNA damage. The rapid activation of p53 by ionizing radiation and radiomimetic agents is largely dependent on the ATM kinase. p53 is phosphorylated by ATM shortly after DNA damage, resulting in enhanced stability and activity of p53. The Mdm2 oncoprotein is a pivotal negative regulator of p53. In response to ionizing radiation and radiomimetic drugs, Mdm2 undergoes rapid ATM-dependent phosphorylation prior to p53 accumulation. This results in a decrease in its reactivity with the 2A10 monoclonal antibody. Phage display analysis identified a consensus 2A10 recognition sequence, possessing the core motif DYS. Unexpectedly, this motif appears twice within the human Mdm2 molecule, at positions corresponding to residues 258–260 and 393–395. Both putative 2A10 epitopes are highly conserved and encompass potential phosphorylation sites. Serine 395, residing within the carboxy-terminal 2A10 epitope, is the major target on Mdm2 for phosphorylation by ATM in vitro. Mutational analysis supports the conclusion that Mdm2 undergoes ATM-dependent phosphorylation on serine 395 in vivo in response to DNA damage. The data further suggests that phosphorylated Mdm2 may be less capable of promoting the nucleo-cytoplasmic shuttling of p53 and its subsequent degradation, thereby enabling p53 accumulation. Our findings imply that activation of p53 by DNA damage is achieved, in part, through attenuation of the p53-inhibitory potential of Mdm2. PMID:11331603

ATM-dependent E2F1 accumulation in the nucleolus is an indicator of ribosomal stress in early response to DNA damage

PubMed Central

Jin, Ya-Qiong; An, Guo-Shun; Ni, Ju-Hua; Li, Shu-Yan; Jia, Hong-Ti

2014-01-01

The nucleolus plays a major role in ribosome biogenesis. Most genotoxic agents disrupt nucleolar structure and function, which results in the stabilization/activation of p53, inducing cell cycle arrest or apoptosis. Likewise, transcription factor E2F1 as a DNA damage responsive protein also plays roles in cell cycle arrest, DNA repair, or apoptosis in response to DNA damage through transcriptional response and protein–protein interaction. Furthermore, E2F1 is known to be involved in regulating rRNA transcription. However, how E2F1 displays in coordinating DNA damage and nucleolar stress is unclear. In this study, we demonstrate that ATM-dependent E2F1 accumulation in the nucleolus is a characteristic feature of nucleolar stress in early response to DNA damage. We found that at the early stage of DNA damage, E2F1 accumulation in the nucleolus was an ATM-dependent and a common event in p53-suficient and -deficient cells. Increased nucleolar E2F1 was sequestered by the nucleolar protein p14ARF, which repressed E2F1-dependent rRNA transcription initiation, and was coupled with S phase. Our data indicate that early accumulation of E2F1 in the nucleolus is an indicator for nucleolar stress and a component of ATM pathway, which presumably buffers elevation of E2F1 in the nucleoplasm and coordinates the diversifying mechanisms of E2F1 acts in cell cycle progression and apoptosis in early response to DNA damage. PMID:24675884

ATM-dependent E2F1 accumulation in the nucleolus is an indicator of ribosomal stress in early response to DNA damage.

PubMed

Jin, Ya-Qiong; An, Guo-Shun; Ni, Ju-Hua; Li, Shu-Yan; Jia, Hong-Ti

2014-01-01

The nucleolus plays a major role in ribosome biogenesis. Most genotoxic agents disrupt nucleolar structure and function, which results in the stabilization/activation of p53, inducing cell cycle arrest or apoptosis. Likewise, transcription factor E2F1 as a DNA damage responsive protein also plays roles in cell cycle arrest, DNA repair, or apoptosis in response to DNA damage through transcriptional response and protein-protein interaction. Furthermore, E2F1 is known to be involved in regulating rRNA transcription. However, how E2F1 displays in coordinating DNA damage and nucleolar stress is unclear. In this study, we demonstrate that ATM-dependent E2F1 accumulation in the nucleolus is a characteristic feature of nucleolar stress in early response to DNA damage. We found that at the early stage of DNA damage, E2F1 accumulation in the nucleolus was an ATM-dependent and a common event in p53-suficient and -deficient cells. Increased nucleolar E2F1 was sequestered by the nucleolar protein p14ARF, which repressed E2F1-dependent rRNA transcription initiation, and was coupled with S phase. Our data indicate that early accumulation of E2F1 in the nucleolus is an indicator for nucleolar stress and a component of ATM pathway, which presumably buffers elevation of E2F1 in the nucleoplasm and coordinates the diversifying mechanisms of E2F1 acts in cell cycle progression and apoptosis in early response to DNA damage.

Tumors overexpressing RNF168 show altered DNA repair and responses to genotoxic treatments, genomic instability and resistance to proteotoxic stress.

PubMed

Chroma, K; Mistrik, M; Moudry, P; Gursky, J; Liptay, M; Strauss, R; Skrott, Z; Vrtel, R; Bartkova, J; Kramara, J; Bartek, J

2017-04-27

Chromatin DNA damage response (DDR) is orchestrated by the E3 ubiquitin ligase ring finger protein 168 (RNF168), resulting in ubiquitin-dependent recruitment of DDR factors and tumor suppressors breast cancer 1 (BRCA1) and p53 binding protein 1 (53BP1). This ubiquitin signaling regulates pathway choice for repair of DNA double-strand breaks (DSB), toxic lesions whose frequency increases during tumorigenesis. Recruitment of 53BP1 curbs DNA end resection, thereby limiting homologous recombination (HR) and directing DSB repair toward error-prone non-homologous end joining (NHEJ). Under cancer-associated ubiquitin starvation conditions reflecting endogenous or treatment-evoked proteotoxic stress, the ubiquitin-dependent accrual of 53BP1 and BRCA1 at the DNA damage sites is attenuated or lost. Challenging this current paradigm, here we identified diverse human cancer cell lines that display 53BP1 recruitment to DSB sites even under proteasome inhibitor-induced proteotoxic stress, that is, under substantial depletion of free ubiquitin. We show that central to this unexpected phenotype is overabundance of RNF168 that enables more efficient exploitation of the residual-free ubiquitin. Cells with elevated RNF168 are more resistant to combined treatment by ionizing radiation and proteasome inhibition, suggesting that such aberrant RNF168-mediated signaling might reflect adaptation to chronic proteotoxic and genotoxic stresses experienced by tumor cells. Moreover, the overabundant RNF168 and the ensuing unorthodox recruitment patterns of 53BP1, RIF1 and REV7 (monitored on laser micro-irradiation-induced DNA damage) shift the DSB repair balance from HR toward NHEJ, a scenario accompanied by enhanced chromosomal instability/micronuclei formation and sensitivity under replication stress-inducing treatments with camptothecin or poly(ADP-ribose) polymerase (PARP) inhibitor. Overall, our data suggest that the deregulated RNF168/53BP1 pathway could promote tumorigenesis by selecting

ATM-Dependent Phosphorylation of MEF2D Promotes Neuronal Survival after DNA Damage

PubMed Central

Chan, Shing Fai; Sances, Sam; Brill, Laurence M.; Okamoto, Shu-ichi; Zaidi, Rameez; McKercher, Scott R.; Akhtar, Mohd W.; Nakanishi, Nobuki

2014-01-01

Mutations in the ataxia telangiectasia mutated (ATM) gene, which encodes a kinase critical for the normal DNA damage response, cause the neurodegenerative disorder ataxia-telangiectasia (AT). The substrates of ATM in the brain are poorly understood. Here we demonstrate that ATM phosphorylates and activates the transcription factor myocyte enhancer factor 2D (MEF2D), which plays a critical role in promoting survival of cerebellar granule cells. ATM associates with MEF2D after DNA damage and phosphorylates the transcription factor at four ATM consensus sites. Knockdown of endogenous MEF2D with a short-hairpin RNA (shRNA) increases sensitivity to etoposide-induced DNA damage and neuronal cell death. Interestingly, substitution of endogenous MEF2D with an shRNA-resistant phosphomimetic MEF2D mutant protects cerebellar granule cells from cell death after DNA damage, whereas an shRNA-resistant nonphosphorylatable MEF2D mutant does not. In vivo, cerebella in Mef2d knock-out mice manifest increased susceptibility to DNA damage. Together, our results show that MEF2D is a substrate for phosphorylation by ATM, thus promoting survival in response to DNA damage. Moreover, dysregulation of the ATM–MEF2D pathway may contribute to neurodegeneration in AT. PMID:24672010

Inhibition of autophagy enhances DNA damage-induced apoptosis by disrupting CHK1-dependent S phase arrest

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liou, Jong-Shian; Wu, Yi-Chen; Yen, Wen-Yen

2014-08-01

DNA damage has been shown to induce autophagy, but the role of autophagy in the DNA damage response and cell fate is not fully understood. BO-1012, a bifunctional alkylating derivative of 3a-aza-cyclopenta[a]indene, is a potent DNA interstrand cross-linking agent with anticancer activity. In this study, BO-1012 was found to reduce DNA synthesis, inhibit S phase progression, and induce phosphorylation of histone H2AX on serine 139 (γH2AX) exclusively in S phase cells. Both CHK1 and CHK2 were phosphorylated in response to BO-1012 treatment, but only depletion of CHK1, but not CHK2, impaired BO-1012-induced S phase arrest and facilitated the entry ofmore » γH2AX-positive cells into G2 phase. CHK1 depletion also significantly enhanced BO-1012-induced cell death and apoptosis. These results indicate that BO-1012-induced S phase arrest is a CHK1-dependent pro-survival response. BO-1012 also resulted in marked induction of acidic vesicular organelle (AVO) formation and microtubule-associated protein 1 light chain 3 (LC3) processing and redistribution, features characteristic of autophagy. Depletion of ATG7 or co-treatment of cells with BO-1012 and either 3-methyladenine or bafilomycin A1, two inhibitors of autophagy, not only reduced CHK1 phosphorylation and disrupted S phase arrest, but also increased cleavage of caspase-9 and PARP, and cell death. These results suggest that cells initiate S phase arrest and autophagy as pro-survival responses to BO-1012-induced DNA damage, and that suppression of autophagy enhances BO-1012-induced apoptosis via disruption of CHK1-dependent S phase arrest. - Highlights: • Autophagy inhibitors enhanced the cytotoxicity of a DNA alkylating agent, BO-1012. • BO-1012-induced S phase arrest was a CHK1-dependent pro-survival response. • Autophagy inhibition enhanced BO-1012 cytotoxicity via disrupting the S phase arrest.« less

Adenovirus Core Protein VII Protects the Viral Genome from a DNA Damage Response at Early Times after Infection▿

PubMed Central

Karen, Kasey A.; Hearing, Patrick

2011-01-01

Adenovirus has a linear, double-stranded DNA genome that is perceived by the cellular Mre11-Rad50-Nbs1 (MRN) DNA repair complex as a double-strand break. If unabated, MRN elicits a double-strand break repair response that blocks viral DNA replication and ligates the viral genomes into concatemers. There are two sets of early viral proteins that inhibit the MRN complex. The E1B-55K/E4-ORF6 complex recruits an E3 ubiquitin ligase and targets MRN proteins for proteasome-dependent degradation. The E4-ORF3 protein inhibits MRN through sequestration. The mechanism that prevents MRN recognition of the viral genome prior to the expression of these early proteins was previously unknown. Here we show a temporal correlation between the loss of viral core protein VII from the adenovirus genome and a gain of checkpoint signaling due to the double-strand break repair response. While checkpoint signaling corresponds to the recognition of the viral genome, core protein VII binding to and checkpoint signaling at viral genomes are largely mutually exclusive. Transcription is known to release protein VII from the genome, and the inhibition of transcription shows a decrease in checkpoint signaling. Finally, we show that the nuclease activity of Mre11 is dispensable for the inhibition of viral DNA replication during a DNA damage response. These results support a model involving the protection of the incoming viral genome from checkpoint signaling by core protein VII and suggest that the induction of an MRN-dependent DNA damage response may inhibit adenovirus replication by physically masking the origins of DNA replication rather than altering their integrity. PMID:21345950

Skeletal muscle and liver contain a soluble ATP + ubiquitin-dependent proteolytic system.

PubMed Central

Fagan, J M; Waxman, L; Goldberg, A L

1987-01-01

Although protein breakdown in most cells seems to require metabolic energy, it has only been possible to establish a soluble ATP-dependent proteolytic system in extracts of reticulocytes and erythroleukemia cells. We have now succeeded in demonstrating in soluble extracts and more purified preparations from rabbit skeletal muscle a 12-fold stimulation by ATP of breakdown of endogenous proteins and a 6-fold stimulation of 125I-lysozyme degradation. However, it has still not been possible to demonstrate such large effects of ATP in similar preparations from liver. Nevertheless, after fractionation by DEAE-chromatography and gel filtration, we found that extracts from liver as well as muscle contain both the enzymes which conjugate ubiquitin to 125I-lysozyme and an enzyme which specifically degrades the ubiquitin-protein conjugates. When this proteolytic activity was recombined with the conjugating enzymes, ATP + ubiquitin-dependent degradation of many proteins was observed. This proteinase is unusually large, approx. 1500 kDa, requires ATP hydrolysis for activity and resembles the ubiquitin-protein-conjugate degrading activity isolated from reticulocytes. Thus the ATP + ubiquitin-dependent pathway is likely to be present in all mammalian cells, although certain tissues may contain inhibitory factors. Images Fig. 2. PMID:2820375

ATM-dependent DNA damage checkpoint functions regulate gene expression in human fibroblasts

PubMed Central

Zhou, Tong; Chou, Jeff; Zhou, Yingchun; Simpson, Dennis A.; Cao, Feng; Bushel, Pierre R.; Paules, Richard S.; Kaufmann, William K.

2013-01-01

The relationships between profiles of global gene expression and DNA damage checkpoint functions were studied in cells from patients with ataxia telangiectasia (AT). Three telomerase-expressing AT fibroblast lines displayed the expected hypersensitivity to ionizing radiation (IR) and defects in DNA damage checkpoints. Profiles of global gene expression in AT cells were determined at 2, 6 and 24 h after treatment with 1.5 Gy IR or sham-treatment, and were compared to those previously recognized in normal human fibroblasts. Under basal conditions 160 genes or ESTs were differentially expressed in AT and normal fibroblasts, and these were associated by gene ontology with insulin-like growth factor binding and regulation of cell growth. Upon DNA damage, 1091 gene mRNAs were changed in at least two of the three AT cell lines. When compared with the 1811 genes changed in normal human fibroblasts after the same treatment, 715 were found in both AT and normal fibroblasts, including most genes categorized by gene ontology into cell cycle, cell growth and DNA damage response pathways. However, the IR-induced changes in these 715 genes in AT cells usually were delayed or attenuated in comparison to normal cells. The reduced change in DNA-damage-response genes and the attenuated repression of cell-cycle-regulated genes may account for the defects in cell cycle checkpoint function in AT cells. PMID:17699107

Replication licensing and the DNA damage checkpoint

PubMed Central

Cook, Jeanette Gowen

2011-01-01

Accurate and timely duplication of chromosomal DNA requires that replication be coordinated with processes that ensure genome integrity. Significant advances in determining how the earliest steps in DNA replication are affected by DNA damage have highlighted some of the mechanisms to establish that coordination. Recent insights have expanded the relationship between the ATM and ATR-dependent checkpoint pathways and the proteins that bind and function at replication origins. These findings suggest that checkpoints and replication are more intimately associated than previously appreciated, even in the absence of exogenous DNA damage. This review summarizes some of these developments. PMID:19482602

DNA damage induced by the direct effect of radiation

NASA Astrophysics Data System (ADS)

Yokoya, A.; Shikazono, N.; Fujii, K.; Urushibara, A.; Akamatsu, K.; Watanabe, R.

2008-10-01

We have studied the nature of DNA damage induced by the direct effect of radiation. The yields of single- (SSB) and double-strand breaks (DSB), base lesions and clustered damage were measured using the agarose gel electrophoresis method after exposing to various kinds of radiations to a simple model DNA molecule, fully hydrated closed-circular plasmid DNA (pUC18). The yield of SSB does not show significant dependence on linear energy transfer (LET) values. On the other hand, the yields of base lesions revealed by enzymatic probes, endonuclease III (Nth) and formamidopyrimidine DNA glycosylase (Fpg), which excise base lesions and leave a nick at the damage site, strongly depend on LET values. Soft X-ray photon (150 kVp) irradiation gives a maximum yield of the base lesions detected by the enzymatic probes as SSB and clustered damage, which is composed of one base lesion and proximate other base lesions or SSBs. The clustered damage is visualized as an enzymatically induced DSB. The yields of the enzymatically additional damages strikingly decrease with increasing levels of LET. These results suggest that in higher LET regions, the repair enzymes used as probes are compromised because of the dense damage clustering. The studies using simple plasmid DNA as a irradiation sample, however, have a technical difficulty to detect multiple SSBs in a plasmid DNA. To detect the additional SSBs induced in opposite strand of the first SSB, we have also developed a novel technique of DNA-denaturation assay. This allows us to detect multiply induced SSBs in both strand of DNA, but not induced DSB.

Age-dependent systemic DNA damage in early Type 2 Diabetes mellitus.

PubMed

Rogulj, Dinko; El Aklouk, Ismail; Konjevoda, Paško; Ljubić, Spomenka; Pibernik Okanović, Mirjana; Barbir, Ante; Luburić, Marijana; Radman, Maja; Budinski, Ninoslav; Vučić Lovrenčić, Marijana

2017-01-01

Oxidative stress, capable of eliciting damage to various biomolecules including DNA, is a recognized component of diabetes mellitus and its complications. Metabolic syndrome (MetS) is associated with the development of type 2 diabetes mellitus (T2DM), as well as other unfavorable outcomes. The aim of this study was to elucidate the role of oxidative stress in the development of T2DM, by investigating association of oxidative DNA damage with metabolic parameters in subjects with MetS and early T2DM. Selected anthropometric and biochemical parameters of MetS, inflammation and oxidative DNA damage: body mass index (BMI), fatty liver index (FLI), waist circumference (WC), total cholesterol, HDL and LDL-cholesterol, gamma-glutamyl transpeptidase (GGT), uric acid, C-reactive protein (CRP), total leukocyte/neutrophil count, and urinary 8-hidroxy-deoxyguanosine (u-8-OHdG) were assessed in male subjects with MetS and both younger (≤55 years) and older (>55 years) subjects with T2DM of short duration without complications. BMI, FLI, WC, total and LDL-cholesterol and uric acid were higher, while the u-8-OHdG was lower in MetS group, when compared to older T2DM subjects. None of these parameters were different neither between MetS and younger T2DM, nor between two sub-groups of subjects with T2DM. Values of CRP, HDL-cholesterol, triglycerides, GGT, leukocytes and neutrophils were not different between all examined groups of subjects. Higher 8-OHdG in older subjects with T2DM suggests that both aging process and diabetes could contribute to the development of DNA damage. Oxidative DNA damage cannot serve as an universal early marker of T2DM.

Mechanism of UVA-dependent DNA damage induced by an antitumor drug dacarbazine in relation to its photogenotoxicity.

PubMed

Iwamoto, Takuya; Hiraku, Yusuke; Okuda, Masahiro; Kawanishi, Shosuke

2008-03-01

It has been reported that dacarbazine (DTIC) is photogenotoxic. The purpose of this study is to clarify the mechanism of photogenotoxicity induced by DTIC. We examined DNA damage induced by UVA-irradiated DTIC using 32P-5'-end-labeled DNA fragments obtained from human genes. Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in calf thymus DNA was measured by high performance liquid chromatograph with an electrochemical detector. Electron spin resonance (ESR) spin-trapping experiments were performed to detect radical species generated from UVA-irradiated DTIC. UVA-irradiated DTIC caused DNA damage at guanine residues, especially at the 5'-GGT-3' sequence in the presence of Cu(II) and also induced 8-oxodG generation in calf thymus DNA. DTIC-induced photodamage to DNA fragments was partially inhibited by catalase, whereas 8-oxodG formation was significantly increased by catalase. NaN3, a carbene scavenger, inhibited DNA damage and 8-oxodG formation in a dose-dependent manner, suggesting that carbene intermediates are involved. The ESR spin-trapping experiments demonstrated the generation of aryl radicals in the process of photodegradation of DTIC. Photoactivated DTIC generates the carbene and aryl radicals, which may induce both DNA adduct and 8-oxodG formation, resulting in photogenotoxicity. This study could provide an insight into the safe usage of DTIC.

Ubiquitin-dependent Regulation of Phospho-AKT Dynamics by the Ubiquitin E3 Ligase, NEDD4-1, in the Insulin-like Growth Factor-1 Response*

PubMed Central

Fan, Chuan-Dong; Lum, Michelle A.; Xu, Chao; Black, Jennifer D.; Wang, Xinjiang

2013-01-01

AKT is a critical effector kinase downstream of the PI3K pathway that regulates a plethora of cellular processes including cell growth, death, differentiation, and migration. Mechanisms underlying activated phospho-AKT (pAKT) translocation to its action sites remain unclear. Here we show that NEDD4-1 is a novel E3 ligase that specifically regulates ubiquitin-dependent trafficking of pAKT in insulin-like growth factor (IGF)-1 signaling. NEDD4-1 physically interacts with AKT and promotes HECT domain-dependent ubiquitination of exogenous and endogenous AKT. NEDD4-1 catalyzes K63-type polyubiquitin chain formation on AKT in vitro. Plasma membrane binding is the key step for AKT ubiquitination by NEDD4-1 in vivo. Ubiquitinated pAKT translocates to perinuclear regions, where it is released into the cytoplasm, imported into the nucleus, or coupled with proteasomal degradation. IGF-1 signaling specifically stimulates NEDD4-1-mediated ubiquitination of pAKT, without altering total AKT ubiquitination. A cancer-derived plasma membrane-philic mutant AKT(E17K) is more effectively ubiquitinated by NEDD4-1 and more efficiently trafficked into the nucleus compared with wild type AKT. This study reveals a novel mechanism by which a specific E3 ligase is required for ubiquitin-dependent control of pAKT dynamics in a ligand-specific manner. PMID:23195959

Terminating protein ubiquitination: Hasta la vista, ubiquitin.

PubMed

Stringer, Daniel K; Piper, Robert C

2011-09-15

Ubiquitination is a post-translational modification that generally directs proteins for degradation by the proteasome or by lysosomes. However, ubiquitination has been implicated in many other cellular processes, including transcriptional regulation, DNA repair, regulation of protein-protein interactions and association with ubiquitin-binding scaffolds. Ubiquitination is a dynamic process. Ubiquitin is added to proteins by E3 ubiquitin ligases as a covalent modification to one or multiple lysine residues as well as non-lysine amino acids. Ubiquitin itself contains seven lysines, each of which can also be ubiquitinated, leading to polyubiquitin chains that are best characterized for linkages occurring through K48 and K63. Ubiquitination can also be reversed by the action of deubiquitination enzymes (DUbs). Like E3 ligases, DUbs play diverse and critical roles in cells. ( 1) Ubiquitin is expressed as a fusion protein, as a linear repeat or as a fusion to ribosomal subunits, and DUbs are necessary to liberate free ubiquitin, making them the first enzyme of the ubiquitin cascade. Proteins destined for degradation by the proteasome or by lysosomes are deubiquitinated prior to their degradation, which allows ubiquitin to be recycled by the cell, contributing to the steady-state pool of free ubiquitin. Proteins destined for degradation by lysosomes are also acted upon by both ligases and DUbs. Deubiquitination can also act as a means to prevent protein degradation, and many proteins are thought to undergo rounds of ubiquitination and deubiquitination, ultimately resulting in either the degradation or stabilization of those proteins. Despite years of study, examining the effects of the ubiquitination of proteins remains quite challenging. This is because the methods that are currently being employed to study ubiquitination are limiting. Here, we briefly examine current strategies to study the effects of ubiquitination and describe an additional novel approach that we have

ATR-dependent phosphorylation of FANCA on serine 1449 after DNA damage is important for FA pathway function

PubMed Central

Collins, Natalie B.; Wilson, James B.; Bush, Thomas; Thomashevski, Andrei; Roberts, Kate J.; Jones, Nigel J.

2009-01-01

Previous work has shown several proteins defective in Fanconi anemia (FA) are phosphorylated in a functionally critical manner. FANCA is phosphorylated after DNA damage and localized to chromatin, but the site and significance of this phosphorylation are unknown. Mass spectrometry of FANCA revealed one phosphopeptide, phosphorylated on serine 1449. Serine 1449 phosphorylation was induced after DNA damage but not during S phase, in contrast to other posttranslational modifications of FA proteins. Furthermore, the S1449A mutant failed to completely correct a variety of FA-associated phenotypes. The DNA damage response is coordinated by phosphorylation events initiated by apical kinases ATM (ataxia telangectasia mutated) and ATR (ATM and Rad3-related), and ATR is essential for proper FA pathway function. Serine 1449 is in a consensus ATM/ATR site, phosphorylation in vivo is dependent on ATR, and ATR phosphorylated FANCA on serine 1449 in vitro. Phosphorylation of FANCA on serine 1449 is a DNA damage–specific event that is downstream of ATR and is functionally important in the FA pathway. PMID:19109555

The budding yeast Rad9 checkpoint protein is subjected to Mec1/Tel1-dependent hyperphosphorylation and interacts with Rad53 after DNA damage.

PubMed

Vialard, J E; Gilbert, C S; Green, C M; Lowndes, N F

1998-10-01

The Saccharomyces cerevisiae RAD9 checkpoint gene is required for transient cell-cycle arrests and transcriptional induction of DNA repair genes in response to DNA damage. Polyclonal antibodies raised against the Rad9 protein recognized several polypeptides in asynchronous cultures, and in cells arrested in S or G2/M phases while a single form was observed in G1-arrested cells. Treatment with various DNA damaging agents, i.e. UV, ionizing radiation or methyl methane sulfonate, resulted in the appearance of hypermodified forms of the protein. All modifications detected during a normal cell cycle and after DNA damage were sensitive to phosphatase treatment, indicating that they resulted from phosphorylation. Damage-induced hyperphosphorylation of Rad9 correlated with checkpoint functions (cell-cycle arrest and transcriptional induction) and was cell-cycle stage- and progression-independent. In asynchronous cultures, Rad9 hyperphosphorylation was dependent on MEC1 and TEL1, homologues of the ATR and ATM genes. In G1-arrested cells, damage-dependent hyperphosphorylation required functional MEC1 in addition to RAD17, RAD24, MEC3 and DDC1, demonstrating cell-cycle stage specificity of the checkpoint genes in this response to DNA damage. Analysis of checkpoint protein interactions after DNA damage revealed that Rad9 physically associates with Rad53.

Ubiquitin-specific Protease 7 Regulates Nucleotide Excision Repair through Deubiquitinating XPC Protein and Preventing XPC Protein from Undergoing Ultraviolet Light-induced and VCP/p97 Protein-regulated Proteolysis*

PubMed Central

He, Jinshan; Zhu, Qianzheng; Wani, Gulzar; Sharma, Nidhi; Han, Chunhua; Qian, Jiang; Pentz, Kyle; Wang, Qi-en; Wani, Altaf A.

2014-01-01

Ubiquitin specific protease 7 (USP7) is a known deubiquitinating enzyme for tumor suppressor p53 and its downstream regulator, E3 ubiquitin ligase Mdm2. Here we report that USP7 regulates nucleotide excision repair (NER) via deubiquitinating xeroderma pigmentosum complementation group C (XPC) protein, a critical damage recognition factor that binds to helix-distorting DNA lesions and initiates NER. XPC is ubiquitinated during the early stage of NER of UV light-induced DNA lesions. We demonstrate that transiently compromising cellular USP7 by siRNA and chemical inhibition leads to accumulation of ubiquitinated forms of XPC, whereas complete USP7 deficiency leads to rapid ubiquitin-mediated XPC degradation upon UV irradiation. We show that USP7 physically interacts with XPC in vitro and in vivo. Overexpression of wild-type USP7, but not its catalytically inactive or interaction-defective mutants, reduces the ubiquitinated forms of XPC. Importantly, USP7 efficiently deubiquitinates XPC-ubiquitin conjugates in deubiquitination assays in vitro. We further show that valosin-containing protein (VCP)/p97 is involved in UV light-induced XPC degradation in USP7-deficient cells. VCP/p97 is readily recruited to DNA damage sites and colocalizes with XPC. Chemical inhibition of the activity of VCP/p97 ATPase causes an increase in ubiquitinated XPC on DNA-damaged chromatin. Moreover, USP7 deficiency severely impairs the repair of cyclobutane pyrimidine dimers and, to a lesser extent, affects the repair of 6-4 photoproducts. Taken together, our findings uncovered an important role of USP7 in regulating NER via deubiquitinating XPC and by preventing its VCP/p97-regulated proteolysis. PMID:25118285

Interplay of space radiation and microgravity in DNA damage and DNA damage response.

PubMed

Moreno-Villanueva, María; Wong, Michael; Lu, Tao; Zhang, Ye; Wu, Honglu

2017-01-01

In space, multiple unique environmental factors, particularly microgravity and space radiation, pose constant threat to the DNA integrity of living organisms. Specifically, space radiation can cause damage to DNA directly, through the interaction of charged particles with the DNA molecules themselves, or indirectly through the production of free radicals. Although organisms have evolved strategies on Earth to confront such damage, space environmental conditions, especially microgravity, can impact DNA repair resulting in accumulation of severe DNA lesions. Ultimately these lesions, namely double strand breaks, chromosome aberrations, micronucleus formation, or mutations, can increase the risk for adverse health effects, such as cancer. How spaceflight factors affect DNA damage and the DNA damage response has been investigated since the early days of the human space program. Over the years, these experiments have been conducted either in space or using ground-based analogs. This review summarizes the evidence for DNA damage induction by space radiation and/or microgravity as well as spaceflight-related impacts on the DNA damage response. The review also discusses the conflicting results from studies aimed at addressing the question of potential synergies between microgravity and radiation with regard to DNA damage and cellular repair processes. We conclude that further experiments need to be performed in the true space environment in order to address this critical question.

A role for nuclear translocation of tripeptidyl-peptidase II in reactive oxygen species-dependent DNA damage responses.

PubMed

Preta, Giulio; de Klark, Rainier; Glas, Rickard

2009-11-27

Responses to DNA damage are influenced by cellular metabolism through the continuous production of reactive oxygen species (ROS), of which most are by-products of mitochondrial respiration. ROS have a strong influence on signaling pathways during responses to DNA damage, by relatively unclear mechanisms. Previous reports have shown conflicting data on a possible role for tripeptidyl-peptidase II (TPPII), a large cytosolic peptidase, within the DNA damage response. Here we show that TPPII translocated into the nucleus in a p160-ROCK-dependent fashion in response to gamma-irradiation, and that nuclear expression of TPPII was present in most gamma-irradiated transformed cell lines. We used a panel of nine cell lines of diverse tissue origin, including four lymphoma cell lines (T, B and Hodgkins lymphoma), a melanoma, a sarcoma, a colon and two breast carcinomas, where seven out of nine cell lines showed nuclear TPPII expression after gamma-irradiation. Further, this required cellular production of ROS; treatment with either N-acetyl-Cysteine (anti-oxidant) or Rotenone (inhibitor of mitochondrial respiration) inhibited nuclear accumulation of TPPII. The local density of cells was important for nuclear accumulation of TPPII at early time-points following gamma-irradiation (at 1-4h), indicating a bystander effect. Further, we showed that the peptide-based inhibitor Z-Gly-Leu-Ala-OH, but not its analogue Z-Gly-(D)-Leu-Ala-OH, excluded TPPII from the nucleus. This correlated with reduced nuclear expression of p53 as well as caspase-3 and -9 activation in gamma-irradiated lymphoma cells. Our data suggest a role for TPPII in ROS-dependent DNA damage responses, through alteration of its localization from the cytosol into the nucleus.

Mitochondrial DNA Damage and its Consequences for Mitochondrial Gene Expression

PubMed Central

Cline, Susan D.

2012-01-01

How mitochondria process DNA damage and whether a change in the steady-state level of mitochondrial DNA damage (mtDNA) contributes to mitochondrial dysfunction are questions that fuel burgeoning areas of research into aging and disease pathogenesis. Over the past decade, researchers have identified and measured various forms of endogenous and environmental mtDNA damage and have elucidated mtDNA repair pathways. Interestingly, mitochondria do not appear to contain the full range of DNA repair mechanisms that operate in the nucleus, although mtDNA contains types of damage that are targets of each nuclear DNA repair pathway. The reduced repair capacity may, in part, explain the high mutation frequency of the mitochondrial chromosome. Since mtDNA replication is dependent on transcription, mtDNA damage may alter mitochondrial gene expression at three levels: by causing DNA polymerase γ nucleotide incorporation errors leading to mutations, by interfering with the priming of mtDNA replication by the mitochondrial RNA polymerase, or by inducing transcriptional mutagenesis or premature transcript termination. This review summarizes our current knowledge of mtDNA damage, its repair, and its effects on mtDNA integrity and gene expression. PMID:22728831

DNA Damage, DNA Repair, Aging, and Neurodegeneration

PubMed Central

Maynard, Scott; Fang, Evandro Fei; Scheibye-Knudsen, Morten; Croteau, Deborah L.; Bohr, Vilhelm A.

2015-01-01

Aging in mammals is accompanied by a progressive atrophy of tissues and organs, and stochastic damage accumulation to the macromolecules DNA, RNA, proteins, and lipids. The sequence of the human genome represents our genetic blueprint, and accumulating evidence suggests that loss of genomic maintenance may causally contribute to aging. Distinct evidence for a role of imperfect DNA repair in aging is that several premature aging syndromes have underlying genetic DNA repair defects. Accumulation of DNA damage may be particularly prevalent in the central nervous system owing to the low DNA repair capacity in postmitotic brain tissue. It is generally believed that the cumulative effects of the deleterious changes that occur in aging, mostly after the reproductive phase, contribute to species-specific rates of aging. In addition to nuclear DNA damage contributions to aging, there is also abundant evidence for a causative link between mitochondrial DNA damage and the major phenotypes associated with aging. Understanding the mechanistic basis for the association of DNA damage and DNA repair with aging and age-related diseases, such as neurodegeneration, would give insight into contravening age-related diseases and promoting a healthy life span. PMID:26385091

The effect of acetaminophen on ubiquitin homeostasis in Saccharomyces cerevisiae.

PubMed

Huseinovic, Angelina; van Leeuwen, Jolanda S; van Welsem, Tibor; Stulemeijer, Iris; van Leeuwen, Fred; Vermeulen, Nico P E; Kooter, Jan M; Vos, J Chris

2017-01-01

Acetaminophen (APAP), although considered a safe drug, is one of the major causes of acute liver failure by overdose, and therapeutic chronic use can cause serious health problems. Although the reactive APAP metabolite N-acetyl-p-benzoquinoneimine (NAPQI) is clearly linked to liver toxicity, toxicity of APAP is also found without drug metabolism of APAP to NAPQI. To get more insight into mechanisms of APAP toxicity, a genome-wide screen in Saccharomyces cerevisiae for APAP-resistant deletion strains was performed. In this screen we identified genes related to the DNA damage response. Next, we investigated the link between genotype and APAP-induced toxicity or resistance by performing a more detailed screen with a library containing mutants of 1522 genes related to nuclear processes, like DNA repair and chromatin remodelling. We identified 233 strains that had an altered growth rate relative to wild type, of which 107 showed increased resistance to APAP and 126 showed increased sensitivity. Gene Ontology analysis identified ubiquitin homeostasis, regulation of transcription of RNA polymerase II genes, and the mitochondria-to-nucleus signalling pathway to be associated with APAP resistance, while histone exchange and modification, and vesicular transport were connected to APAP sensitivity. Indeed, we observed a link between ubiquitin levels and APAP resistance, whereby ubiquitin deficiency conferred resistance to APAP toxicity while ubiquitin overexpression resulted in sensitivity. The toxicity profile of various chemicals, APAP, and its positional isomer AMAP on a series of deletion strains with ubiquitin deficiency showed a unique resistance pattern for APAP. Furthermore, exposure to APAP increased the level of free ubiquitin and influenced the ubiquitination of proteins. Together, these results uncover a role for ubiquitin homeostasis in APAP-induced toxicity.

UDP-glucuronosyltransferase-dependent bioactivation of clofibric acid to a DNA-damaging intermediate in mouse hepatocytes.

PubMed

Ghaoui, Roula; Sallustio, Benedetta C; Burcham, Philip C; Fontaine, Frank R

2003-05-06

Glucuronidation of a number of carboxyl-containing drugs generates reactive acyl glucuronide metabolites. These electrophilic species alkylate cell proteins and may be implicated in the pathogenesis of a number of toxic syndromes seen in patients receiving the parent aglycones. Whether acyl glucuronides also attack nuclear DNA is unknown, although the acyl glucuronide formed from clofibric acid was recently found to decrease the transfection efficiency of phage DNA and generate strand breaks in plasmid DNA in vitro. To determine if such a DNA damage occurs within a cellular environment, the comet assay (i.e. single-cell gel electrophoresis) was used to detect DNA lesions in the nuclear genome of isolated mouse hepatocytes cultured with clofibric acid. Overnight exposure to 50 microM and higher concentrations of clofibric acid produced concentration-dependent increases in the comet areas of hepatocyte nuclei, with 1 mM clofibrate producing a 3.6-fold elevation over controls. These effects closely coincided with culture medium concentrations of the glucuronide metabolite formed from clofibric acid, 1-O-beta-clofibryl glucuronide. Consistent with a role for glucuronidation in the DNA damage observed, the glucuronidation inhibitor borneol diminished glucuronide formation from 100 microM clofibrate by 98% and returned comet areas to baseline levels. Collectively, these results suggest that the acyl glucuronide formed from clofibric acid is capable of migrating from its site of formation within the endoplasmic reticulum to generate strand nicks in nuclear DNA.

Endogenous c-Myc is essential for p53-induced apoptosis in response to DNA damage in vivo.

PubMed

Phesse, T J; Myant, K B; Cole, A M; Ridgway, R A; Pearson, H; Muncan, V; van den Brink, G R; Vousden, K H; Sears, R; Vassilev, L T; Clarke, A R; Sansom, O J

2014-06-01

Recent studies have suggested that C-MYC may be an excellent therapeutic cancer target and a number of new agents targeting C-MYC are in preclinical development. Given most therapeutic regimes would combine C-MYC inhibition with genotoxic damage, it is important to assess the importance of C-MYC function for DNA damage signalling in vivo. In this study, we have conditionally deleted the c-Myc gene in the adult murine intestine and investigated the apoptotic response of intestinal enterocytes to DNA damage. Remarkably, c-Myc deletion completely abrogated the immediate wave of apoptosis following both ionizing irradiation and cisplatin treatment, recapitulating the phenotype of p53 deficiency in the intestine. Consistent with this, c-Myc-deficient intestinal enterocytes did not upregulate p53. Mechanistically, this was linked to an upregulation of the E3 Ubiquitin ligase Mdm2, which targets p53 for degradation in c-Myc-deficient intestinal enterocytes. Further, low level overexpression of c-Myc, which does not impact on basal levels of apoptosis, elicited sustained apoptosis in response to DNA damage, suggesting c-Myc activity acts as a crucial cell survival rheostat following DNA damage. We also identify the importance of MYC during DNA damage-induced apoptosis in several other tissues, including the thymus and spleen, using systemic deletion of c-Myc throughout the adult mouse. Together, we have elucidated for the first time in vivo an essential role for endogenous c-Myc in signalling DNA damage-induced apoptosis through the control of the p53 tumour suppressor protein.

XPD-dependent activation of apoptosis in response to triplex-induced DNA damage

PubMed Central

Kaushik Tiwari, Meetu; Rogers, Faye A.

2013-01-01

DNA sequences capable of forming triplexes are prevalent in the human genome and have been found to be intrinsically mutagenic. Consequently, a balance between DNA repair and apoptosis is critical to counteract their effect on genomic integrity. Using triplex-forming oligonucleotides to synthetically create altered helical distortions, we have determined that pro-apoptotic pathways are activated by the formation of triplex structures. Moreover, the TFIIH factor, XPD, occupies a central role in triggering apoptosis in response to triplex-induced DNA strand breaks. Here, we show that triplexes are capable of inducing XPD-independent double strand breaks, which result in the formation of γH2AX foci. XPD was subsequently recruited to the triplex-induced double strand breaks and co-localized with γH2AX at the damage site. Furthermore, phosphorylation of H2AX tyrosine 142 was found to stimulate the signaling pathway of XPD-dependent apoptosis. We suggest that this mechanism may play an active role in minimizing genomic instability induced by naturally occurring noncanonical structures, perhaps protecting against cancer initiation. PMID:23913414

DNA-damage response during mitosis induces whole-chromosome missegregation.

PubMed

Bakhoum, Samuel F; Kabeche, Lilian; Murnane, John P; Zaki, Bassem I; Compton, Duane A

2014-11-01

Many cancers display both structural (s-CIN) and numerical (w-CIN) chromosomal instabilities. Defective chromosome segregation during mitosis has been shown to cause DNA damage that induces structural rearrangements of chromosomes (s-CIN). In contrast, whether DNA damage can disrupt mitotic processes to generate whole chromosomal instability (w-CIN) is unknown. Here, we show that activation of the DNA-damage response (DDR) during mitosis selectively stabilizes kinetochore-microtubule (k-MT) attachments to chromosomes through Aurora-A and PLK1 kinases, thereby increasing the frequency of lagging chromosomes during anaphase. Inhibition of DDR proteins, ATM or CHK2, abolishes the effect of DNA damage on k-MTs and chromosome segregation, whereas activation of the DDR in the absence of DNA damage is sufficient to induce chromosome segregation errors. Finally, inhibiting the DDR during mitosis in cancer cells with persistent DNA damage suppresses inherent chromosome segregation defects. Thus, the DDR during mitosis inappropriately stabilizes k-MTs, creating a link between s-CIN and w-CIN. The genome-protective role of the DDR depends on its ability to delay cell division until damaged DNA can be fully repaired. Here, we show that when DNA damage is induced during mitosis, the DDR unexpectedly induces errors in the segregation of entire chromosomes, thus linking structural and numerical chromosomal instabilities. ©2014 American Association for Cancer Research.

Non-covalent Interactions with SUMO and Ubiquitin Orchestrate Distinct Functions of the SLX4 Complex in Genome Maintenance

PubMed Central

Ouyang, Jian; Garner, Elizabeth; Hallet, Alexander; Nguyen, Hai Dang; Rickman, Kimberly A.; Gill, Grace; Smogorzewska, Agata; Zou, Lee

2014-01-01

SLX4, a coordinator of multiple DNA structure-specific endonucleases, is important for several DNA repair pathways. Non-covalent interactions of SLX4 with ubiquitin are required for localizing SLX4 to DNA-interstrand crosslinks (ICLs), yet how SLX4 is targeted to other functional contexts remains unclear. Here, we show that SLX4 binds SUMO-2/3 chains via SUMO-interacting motifs (SIMs). The SIMs of SLX4 are dispensable for ICL repair, but important for processing CPT-induced replication intermediates, suppressing fragile site instability, and localizing SLX4 to ALT telomeres. The localization of SLX4 to laser-induced DNA damage also requires the SIMs, as well as DNA-end resection, UBC9 and MDC1. Furthermore, the SUMO binding of SLX4 enhances its interaction with specific DNA-damage sensors or telomere-binding proteins, including RPA, MRE11-RAD50-NBS1 and TRF2. Thus, the interactions of SLX4 with SUMO and ubiquitin increase its affinity for factors recognizing different DNA lesions or telomeres, helping to direct the SLX4 complex in distinct functional contexts. PMID:25533185

A role for nuclear translocation of tripeptidyl-peptidase II in reactive oxygen species-dependent DNA damage responses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Preta, Giulio; Klark, Rainier de; Glas, Rickard, E-mail: rickard.glas@ki.se

2009-11-27

Responses to DNA damage are influenced by cellular metabolism through the continuous production of reactive oxygen species (ROS), of which most are by-products of mitochondrial respiration. ROS have a strong influence on signaling pathways during responses to DNA damage, by relatively unclear mechanisms. Previous reports have shown conflicting data on a possible role for tripeptidyl-peptidase II (TPPII), a large cytosolic peptidase, within the DNA damage response. Here we show that TPPII translocated into the nucleus in a p160-ROCK-dependent fashion in response to {gamma}-irradiation, and that nuclear expression of TPPII was present in most {gamma}-irradiated transformed cell lines. We used amore » panel of nine cell lines of diverse tissue origin, including four lymphoma cell lines (T, B and Hodgkins lymphoma), a melanoma, a sarcoma, a colon and two breast carcinomas, where seven out of nine cell lines showed nuclear TPPII expression after {gamma}-irradiation. Further, this required cellular production of ROS; treatment with either N-acetyl-Cysteine (anti-oxidant) or Rotenone (inhibitor of mitochondrial respiration) inhibited nuclear accumulation of TPPII. The local density of cells was important for nuclear accumulation of TPPII at early time-points following {gamma}-irradiation (at 1-4 h), indicating a bystander effect. Further, we showed that the peptide-based inhibitor Z-Gly-Leu-Ala-OH, but not its analogue Z-Gly-(D)-Leu-Ala-OH, excluded TPPII from the nucleus. This correlated with reduced nuclear expression of p53 as well as caspase-3 and -9 activation in {gamma}-irradiated lymphoma cells. Our data suggest a role for TPPII in ROS-dependent DNA damage responses, through alteration of its localization from the cytosol into the nucleus.« less

Base Excision Repair and Lesion-Dependent Subpathways for Repair of Oxidative DNA Damage

PubMed Central

Svilar, David; Goellner, Eva M.; Almeida, Karen H.

2011-01-01

Abstract Nuclear and mitochondrial genomes are under continuous assault by a combination of environmentally and endogenously derived reactive oxygen species, inducing the formation and accumulation of mutagenic, toxic, and/or genome-destabilizing DNA lesions. Failure to resolve these lesions through one or more DNA-repair processes is associated with genome instability, mitochondrial dysfunction, neurodegeneration, inflammation, aging, and cancer, emphasizing the importance of characterizing the pathways and proteins involved in the repair of oxidative DNA damage. This review focuses on the repair of oxidative damage–induced lesions in nuclear and mitochondrial DNA mediated by the base excision repair (BER) pathway in mammalian cells. We discuss the multiple BER subpathways that are initiated by one of 11 different DNA glycosylases of three subtypes: (a) bifunctional with an associated β-lyase activity; (b) monofunctional; and (c) bifunctional with an associated β,δ-lyase activity. These three subtypes of DNA glycosylases all initiate BER but yield different chemical intermediates and hence different BER complexes to complete repair. Additionally, we briefly summarize alternate repair events mediated by BER proteins and the role of BER in the repair of mitochondrial DNA damage induced by ROS. Finally, we discuss the relation of BER and oxidative DNA damage in the onset of human disease. Antioxid. Redox Signal. 14, 2491–2507. PMID:20649466

Cell fate determination by ubiquitin-dependent regulation of translation

PubMed Central

Werner, Achim; Iwasaki, Shintaro; McGourty, Colleen; Medina-Ruiz, Sofia; Teerikorpi, Nia; Fedrigo, Indro; Ingolia, Nicholas T.; Rape, Michael

2015-01-01

Metazoan development depends on accurate execution of differentiation programs that allow pluripotent stem cells to adopt specific fates 1. Differentiation requires changes to chromatin architecture and transcriptional networks, yet whether other regulatory events support cell fate determination is less well understood. Here, we have identified the vertebrate-specific ubiquitin ligase CUL3KBTBD8 as an essential regulator of neural crest specification. CUL3KBTBD8 monoubiquitylates NOLC1 and its paralog TCOF1, whose mutation underlies the neurocristopathy Treacher Collins Syndrome 2,3. Ubiquitylation drives formation of a TCOF1-NOLC1 platform that connects RNA polymerase I with ribosome modification enzymes and remodels the translational program of differentiating cells in favor of neural crest specification. We conclude that ubiquitin-dependent regulation of translation is an important feature of cell fate determination. PMID:26399832

Blood micronutrients and DNA damage in children.

PubMed

Milne, Elizabeth; Greenop, Kathryn R; Ramankutty, Padmaja; Miller, Margaret; de Klerk, Nicholas H; Armstrong, Bruce K; Almond, Theodora; O'Callaghan, Nathan J; Fenech, Michael

2015-10-01

Maintenance of normal cellular phenotype depends largely on accurate DNA replication and repair. DNA damage causes gene mutations and predisposes to cancer and other chronic diseases. Growing evidence indicates that nutritional factors are associated with DNA damage in adults; here, we investigate these associations in children. We conducted a cross-sectional study among 462 healthy children 3, 6, and 9 years of age. Blood was collected and micronutrient levels were measured. The cytokinesis-block micronucleus cytome assay was used to measure chromosomal DNA damage (micronuclei, nucleoplasmic bridges, and nuclear buds) in lymphocytes. Cell apoptosis, necrosis, and the nuclear division index were also measured. Nine loci in genes involved in folate metabolism and DNA repair were genotyped. Data were analyzed using linear regression with adjustment for potential confounders. Plasma calcium was positively associated with micronuclei and necrosis, and α-tocopherol negatively associated with apoptosis, nuclear division index, and nucleoplasmic bridges; lutein was positively associated with nucleoplasmic bridges. α-tocopherol was positively associated with necrosis. DNA damage in healthy children may be influenced by blood micronutrient levels and certain genotypes. Further investigation of associations between nutritional status and genomic integrity in children is needed to shed additional light on potential mechanisms. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DNA damage may drive nucleosomal reorganization to facilitate damage detection

NASA Astrophysics Data System (ADS)

LeGresley, Sarah E.; Wilt, Jamie; Antonik, Matthew

2014-03-01

One issue in genome maintenance is how DNA repair proteins find lesions at rates that seem to exceed diffusion-limited search rates. We propose a phenomenon where DNA damage induces nucleosomal rearrangements which move lesions to potential rendezvous points in the chromatin structure. These rendezvous points are the dyad and the linker DNA between histones, positions in the chromatin which are more likely to be accessible by repair proteins engaged in a random search. The feasibility of this mechanism is tested by considering the statistical mechanics of DNA containing a single lesion wrapped onto the nucleosome. We consider lesions which make the DNA either more flexible or more rigid by modeling the lesion as either a decrease or an increase in the bending energy. We include this energy in a partition function model of nucleosome breathing. Our results indicate that the steady state for a breathing nucleosome will most likely position the lesion at the dyad or in the linker, depending on the energy of the lesion. A role for DNA binding proteins and chromatin remodelers is suggested based on their ability to alter the mechanical properties of the DNA and DNA-histone binding, respectively. We speculate that these positions around the nucleosome potentially serve as rendezvous points where DNA lesions may be encountered by repair proteins which may be sterically hindered from searching the rest of the nucleosomal DNA. The strength of the repositioning is strongly dependent on the structural details of the DNA lesion and the wrapping and breathing of the nucleosome. A more sophisticated evaluation of this proposed mechanism will require detailed information about breathing dynamics, the structure of partially wrapped nucleosomes, and the structural properties of damaged DNA.

Mitochondrial DNA Damage and Diseases.

PubMed

Singh, Gyanesh; Pachouri, U C; Khaidem, Devika Chanu; Kundu, Aman; Chopra, Chirag; Singh, Pushplata

2015-01-01

Various endogenous and environmental factors can cause mitochondrial DNA (mtDNA) damage.  One of the reasons for enhanced mtDNA damage could be its proximity to the source of oxidants, and lack of histone-like protective proteins. Moreover, mitochondria contain inadequate DNA repair pathways, and, diminished DNA repair capacity may be one of the factors responsible for high mutation frequency of the mtDNA. mtDNA damage might cause impaired mitochondrial function, and, unrepaired mtDNA damage has been frequently linked with several diseases. Exploration of mitochondrial perspective of diseases might lead to a better understanding of several diseases, and will certainly open new avenues for detection, cure, and prevention of ailments.

Mitochondrial DNA Damage and Diseases

PubMed Central

Singh, Gyanesh; Pachouri, U C; Khaidem, Devika Chanu; Kundu, Aman; Chopra, Chirag; Singh, Pushplata

2015-01-01

Various endogenous and environmental factors can cause mitochondrial DNA (mtDNA) damage.  One of the reasons for enhanced mtDNA damage could be its proximity to the source of oxidants, and lack of histone-like protective proteins. Moreover, mitochondria contain inadequate DNA repair pathways, and, diminished DNA repair capacity may be one of the factors responsible for high mutation frequency of the mtDNA. mtDNA damage might cause impaired mitochondrial function, and, unrepaired mtDNA damage has been frequently linked with several diseases. Exploration of mitochondrial perspective of diseases might lead to a better understanding of several diseases, and will certainly open new avenues for detection, cure, and prevention of ailments. PMID:27508052

[UV-induced DNA damage and protective effects of antioxidants on DNA damage in human lens epithelial cells studied with comet assay].

PubMed

Wu, Zhi-hong; Wang, Mian-rong; Yan, Qi-chang; Pu, Wei; Zhang, Jin-song

2006-11-01

To investigate the mechanism of UV-induced DNA damage and repair and the protective effects of antioxidants on DNA damage in human lens epithelial cells. Human lens epithelial cells were irradiated at UV-doses 0.0 (control group), 2.5, 5.0, 7.5, 10.0 mJ/cm(2) (treated group 1 - 4). The amounts of DNA single strand breaks (SSB) were measured with the alkaline comet assay (CA). The spontaneous repair of DNA SSB after exposure to UV at 10.0 mJ/cm(2) was also determined in human lens epithelial cells. Human lens epithelial cells were treated with different concentration of VitaminC (VitC), taurine, superoxide dismutase (SOD) and epigallocatechin gallate (EGCG) before and after ultraviolet radiation, the effects of antioxidants on DNA damage was examined with alkaline comet assay. The amount of DNA SSB in control group and treated groups 1 - 4 showed increased tendency, was dose-dependent to the dose of UV irradiation, the differences of DNA SSB in 5 group were significantly (P < 0.01). UV-induced DNA SSB at 10.0 mJ/cm(2) in human lens epithelial cells, the half repair time was 60 minutes. Human lens epithelial cells were treated with different concentrations of taurine, SOD and EGCG before ultraviolet radiation. The differences of DNA damage in control and various antioxidant treated groups was statistically significant (F = 6.591, 13.542, 4.626 in cells treated with taurine, SOD and EGCG, respectively, P < 0.01), the difference of VitC effect on DNA in control and treated group were not significantly (F = 1.451, P > 0.05). Human lens epithelial cells were treated with different concentration of VitC, taurine, SOD and EGCG after ultraviolet radiation. The differences of DNA damage between the control and treated group were statistically significant (F = 6.571, 4.810, 6.824, 9.182 in cells treated with VitC, taurine, SOD and EGCG, respectively, P < 0.01). The differences of protective effects on DNA damage in these four different kinds of antioxidants added before UV

Shape-dependent bactericidal activity of copper oxide nanoparticle mediated by DNA and membrane damage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Laha, Dipranjan; Pramanik, Arindam; Laskar, Aparna

Highlights: • Spherical and sheet shaped copper oxide nanoparticles were synthesized. • Physical characterizations of these nanoparticles were done by TEM, DLS, XRD, FTIR. • They showed shape dependent antibacterial activity on different bacterial strain. • They induced both membrane damage and ROS mediated DNA damage in bacteria. - Abstract: In this work, we synthesized spherical and sheet shaped copper oxide nanoparticles and their physical characterizations were done by the X-ray diffraction, fourier transform infrared spectroscopy, transmission electron microscopy and dynamic light scattering. The antibacterial activity of these nanoparticles was determined on both gram positive and gram negative bacterial. Sphericalmore » shaped copper oxide nanoparticles showed more antibacterial property on gram positive bacteria where as sheet shaped copper oxide nanoparticles are more active on gram negative bacteria. We also demonstrated that copper oxide nanoparticles produced reactive oxygen species in both gram negative and gram positive bacteria. Furthermore, they induced membrane damage as determined by atomic force microscopy and scanning electron microscopy. Thus production of and membrane damage are major mechanisms of the bactericidal activity of these copper oxide nanoparticles. Finally it was concluded that antibacterial activity of nanoparticles depend on physicochemical properties of copper oxide nanoparticles and bacterial strain.« less

Ubiquitin-Fused and/or Multiple Early Genes from Cottontail Rabbit Papillomavirus as DNA Vaccines

PubMed Central

Leachman, Sancy A.; Shylankevich, Mark; Slade, Martin D.; Levine, Dana; K. Sundaram, Ranjini; Xiao, Wei; Bryan, Marianne; Zelterman, Daniel; Tiegelaar, Robert E.; Brandsma, Janet L.

2002-01-01

Human papillomavirus (HPV) vaccines have the potential to prevent cervical cancer by preventing HPV infection or treating premalignant disease. We previously showed that DNA vaccination with the cottontail rabbit papillomavirus (CRPV) E6 gene induced partial protection against CRPV challenge and that the vaccine's effects were greatly enhanced by priming with granulocyte-macrophage colony-stimulating factor (GM-CSF). In the present study, two additional strategies for augmenting the clinical efficacy of CRPV E6 vaccination were evaluated. The first was to fuse a ubiquitin monomer to the CRPV E6 protein to enhance antigen processing and presentation through the major histocompatibility complex class I pathway. Rabbits vaccinated with the wild-type E6 gene plus GM-CSF or with the ubiquitin-fused E6 gene formed significantly fewer papillomas than the controls. The papillomas also required a longer time to appear and grew more slowly. Finally, a significant proportion of the papillomas subsequently regressed. The ubiquitin-fused E6 vaccine was significantly more effective than the wild-type E6 vaccine plus GM-CSF priming. The second strategy was to vaccinate with multiple CRPV early genes to increase the breadth of the CRPV-specific response. DNA vaccines encoding the wild-type CRPV E1-E2, E6, or E7 protein were tested alone and in all possible combinations. All vaccines and combinations suppressed papilloma formation, slowed papilloma growth, and stimulated subsequent papilloma regression. Finally, the two strategies were merged and a combination DNA vaccine containing ubiquitin-fused versions of the CRPV E1, E2, and E7 genes was tested. This last vaccine prevented papilloma formation at all challenge sites in all rabbits, demonstrating complete protection. PMID:12097575

The effect of acetaminophen on ubiquitin homeostasis in Saccharomyces cerevisiae

PubMed Central

Huseinovic, Angelina; van Leeuwen, Jolanda S.; van Welsem, Tibor; Stulemeijer, Iris; van Leeuwen, Fred; Vermeulen, Nico P. E.; Kooter, Jan M.; Vos, J. Chris

2017-01-01

Acetaminophen (APAP), although considered a safe drug, is one of the major causes of acute liver failure by overdose, and therapeutic chronic use can cause serious health problems. Although the reactive APAP metabolite N-acetyl-p-benzoquinoneimine (NAPQI) is clearly linked to liver toxicity, toxicity of APAP is also found without drug metabolism of APAP to NAPQI. To get more insight into mechanisms of APAP toxicity, a genome-wide screen in Saccharomyces cerevisiae for APAP-resistant deletion strains was performed. In this screen we identified genes related to the DNA damage response. Next, we investigated the link between genotype and APAP-induced toxicity or resistance by performing a more detailed screen with a library containing mutants of 1522 genes related to nuclear processes, like DNA repair and chromatin remodelling. We identified 233 strains that had an altered growth rate relative to wild type, of which 107 showed increased resistance to APAP and 126 showed increased sensitivity. Gene Ontology analysis identified ubiquitin homeostasis, regulation of transcription of RNA polymerase II genes, and the mitochondria-to-nucleus signalling pathway to be associated with APAP resistance, while histone exchange and modification, and vesicular transport were connected to APAP sensitivity. Indeed, we observed a link between ubiquitin levels and APAP resistance, whereby ubiquitin deficiency conferred resistance to APAP toxicity while ubiquitin overexpression resulted in sensitivity. The toxicity profile of various chemicals, APAP, and its positional isomer AMAP on a series of deletion strains with ubiquitin deficiency showed a unique resistance pattern for APAP. Furthermore, exposure to APAP increased the level of free ubiquitin and influenced the ubiquitination of proteins. Together, these results uncover a role for ubiquitin homeostasis in APAP-induced toxicity. PMID:28291796

Site-specific Interaction Mapping of Phosphorylated Ubiquitin to Uncover Parkin Activation*♦

PubMed Central

Yamano, Koji; Queliconi, Bruno B.; Koyano, Fumika; Saeki, Yasushi; Hirokawa, Takatsugu; Tanaka, Keiji; Matsuda, Noriyuki

2015-01-01

Damaged mitochondria are eliminated through autophagy machinery. A cytosolic E3 ubiquitin ligase Parkin, a gene product mutated in familial Parkinsonism, is essential for this pathway. Recent progress has revealed that phosphorylation of both Parkin and ubiquitin at Ser65 by PINK1 are crucial for activation and recruitment of Parkin to the damaged mitochondria. However, the mechanism by which phosphorylated ubiquitin associates with and activates phosphorylated Parkin E3 ligase activity remains largely unknown. Here, we analyze interactions between phosphorylated forms of both Parkin and ubiquitin at a spatial resolution of the amino acid residue by site-specific photo-crosslinking. We reveal that the in-between-RING (IBR) domain along with RING1 domain of Parkin preferentially binds to ubiquitin in a phosphorylation-dependent manner. Furthermore, another approach, the Fluoppi (fluorescent-based technology detecting protein-protein interaction) assay, also showed that pathogenic mutations in these domains blocked interactions with phosphomimetic ubiquitin in mammalian cells. Molecular modeling based on the site-specific photo-crosslinking interaction map combined with mass spectrometry strongly suggests that a novel binding mechanism between Parkin and ubiquitin leads to a Parkin conformational change with subsequent activation of Parkin E3 ligase activity. PMID:26260794

PRAP1 is a novel executor of p53-dependent mechanisms in cell survival after DNA damage

PubMed Central

Huang, B H; Zhuo, J L; Leung, C H W; Lu, G D; Liu, J J; Yap, C T; Hooi, S C

2012-01-01

p53 has a crucial role in governing cellular mechanisms in response to a broad range of genotoxic stresses. During DNA damage, p53 can either promote cell survival by activating senescence or cell-cycle arrest and DNA repair to maintain genomic integrity for cell survival or direct cells to undergo apoptosis to eliminate extensively damaged cells. The ability of p53 to execute these two opposing cell fates depends on distinct signaling pathways downstream of p53. In this study, we showed that under DNA damage conditions induced by chemotherapeutic drugs, gamma irradiation and hydrogen peroxide, p53 upregulates a novel protein, proline-rich acidic protein 1 (PRAP1). We identified functional p53-response elements within intron 1 of PRAP1 gene and showed that these regions interact directly with p53 using ChIP assays, indicating that PRAP1 is a novel p53 target gene. The induction of PRAP1 expression by p53 may promote resistance of cancer cells to chemotherapeutic drugs such as 5-fluorouracil (5-FU), as knockdown of PRAP1 increases apoptosis in cancer cells after 5-FU treatment. PRAP1 appears to protect cells from apoptosis by inducing cell-cycle arrest, suggesting that the induction of PRAP1 expression by p53 in response to DNA-damaging agents contributes to cancer cell survival. Our findings provide a greater insight into the mechanisms underlying the pro-survival role of p53 in response to cytotoxic treatments. PMID:23235459

PRAP1 is a novel executor of p53-dependent mechanisms in cell survival after DNA damage.

PubMed

Huang, B H; Zhuo, J L; Leung, C H W; Lu, G D; Liu, J J; Yap, C T; Hooi, S C

2012-12-13

p53 has a crucial role in governing cellular mechanisms in response to a broad range of genotoxic stresses. During DNA damage, p53 can either promote cell survival by activating senescence or cell-cycle arrest and DNA repair to maintain genomic integrity for cell survival or direct cells to undergo apoptosis to eliminate extensively damaged cells. The ability of p53 to execute these two opposing cell fates depends on distinct signaling pathways downstream of p53. In this study, we showed that under DNA damage conditions induced by chemotherapeutic drugs, gamma irradiation and hydrogen peroxide, p53 upregulates a novel protein, proline-rich acidic protein 1 (PRAP1). We identified functional p53-response elements within intron 1 of PRAP1 gene and showed that these regions interact directly with p53 using ChIP assays, indicating that PRAP1 is a novel p53 target gene. The induction of PRAP1 expression by p53 may promote resistance of cancer cells to chemotherapeutic drugs such as 5-fluorouracil (5-FU), as knockdown of PRAP1 increases apoptosis in cancer cells after 5-FU treatment. PRAP1 appears to protect cells from apoptosis by inducing cell-cycle arrest, suggesting that the induction of PRAP1 expression by p53 in response to DNA-damaging agents contributes to cancer cell survival. Our findings provide a greater insight into the mechanisms underlying the pro-survival role of p53 in response to cytotoxic treatments.

Monitoring regulation of DNA repair activities of cultured cells in-gel using the comet assay

PubMed Central

Nickson, Catherine M.; Parsons, Jason L.

2014-01-01

Base excision repair (BER) is the predominant cellular mechanism by which human cells repair DNA base damage, sites of base loss, and DNA single strand breaks of various complexity, that are generated in their thousands in every human cell per day as a consequence of cellular metabolism and exogenous agents, including ionizing radiation. Over the last three decades the comet assay has been employed in scientific research to examine the cellular response to these types of DNA damage in cultured cells, therefore revealing the efficiency and capacity of BER. We have recently pioneered new research demonstrating an important role for post-translational modifications (particularly ubiquitylation) in the regulation of cellular levels of BER proteins, and that subtle changes (∼20–50%) in protein levels following siRNA knockdown of E3 ubiquitin ligases or deubiquitylation enzymes can manifest in significant changes in DNA repair capacity monitored using the comet assay. For example, we have shown that the E3 ubiquitin ligase Mule, the tumor suppressor protein ARF, and the deubiquitylation enzyme USP47 modulate DNA repair by controlling cellular levels of DNA polymerase β, and also that polynucleotide kinase phosphatase levels are controlled by ATM-dependant phosphorylation and Cul4A–DDB1–STRAP-dependent ubiquitylation. In these studies we employed a modification of the comet assay whereby cultured cells, following DNA damage treatment, are embedded in agarose and allowed to repair in-gel prior to lysis and electrophoresis. Whilst this method does have its limitations, it avoids the extensive cell culture-based processing associated with the traditional approach using attached cells and also allows for the examination of much more precise DNA repair kinetics. In this review we will describe, using this modified comet assay, our accumulating evidence that ubiquitylation-dependant regulation of BER proteins has important consequences for overall cellular DNA repair

Loss of p53 induces M-phase retardation following G2 DNA damage checkpoint abrogation.

PubMed

Minemoto, Yuzuru; Uchida, Sanae; Ohtsubo, Motoaki; Shimura, Mari; Sasagawa, Toshiyuki; Hirata, Masato; Nakagama, Hitoshi; Ishizaka, Yukihito; Yamashita, Katsumi

2003-04-01

Most cell lines that lack functional p53 protein are arrested in the G2 phase of the cell cycle due to DNA damage. When the G2 checkpoint is abrogated, these cells are forced into mitotic catastrophe. A549 lung adenocarcinoma cells, in which p53 was eliminated with the HPV16 E6 gene, exhibited efficient arrest in the G2 phase when treated with adriamycin. Administration of caffeine to G2-arrested cells induced a drastic change in cell phenotype, the nature of which depended on the status of p53. Flow cytometric and microscopic observations revealed that cells that either contained or lacked p53 resumed their cell cycles and entered mitosis upon caffeine treatment. However, transit to the M phase was slower in p53-negative cells than in p53-positive cells. Consistent with these observations, CDK1 activity was maintained at high levels, along with stable cyclin B1, in p53-negative cells. The addition of butyrolactone I, which is an inhibitor of CDK1 and CDK2, to the p53-negative cells reduced the floating round cell population and induced the disappearance of cyclin B1. These results suggest a relationship between the p53 pathway and the ubiquitin-mediated degradation of mitotic cyclins and possible cross-talk between the G2-DNA damage checkpoint and the mitotic checkpoint.

PIP degron proteins, substrates of CRL4Cdt2, and not PIP boxes, interfere with DNA polymerase η and κ focus formation on UV damage

PubMed Central

Tsanov, Nikolay; Kermi, Chames; Coulombe, Philippe; Van der Laan, Siem; Hodroj, Dana; Maiorano, Domenico

2014-01-01

Proliferating cell nuclear antigen (PCNA) is a well-known scaffold for many DNA replication and repair proteins, but how the switch between partners is regulated is currently unclear. Interaction with PCNA occurs via a domain known as a PCNA-Interacting Protein motif (PIP box). More recently, an additional specialized PIP box has been described, the « PIP degron », that targets PCNA-interacting proteins for proteasomal degradation via the E3 ubiquitin ligase CRL4Cdt2. Here we provide evidence that CRL4Cdt2-dependent degradation of PIP degron proteins plays a role in the switch of PCNA partners during the DNA damage response by facilitating accumulation of translesion synthesis DNA polymerases into nuclear foci. We show that expression of a nondegradable PIP degron (Cdt1) impairs both Pol η and Pol κ focus formation on ultraviolet irradiation and reduces cell viability, while canonical PIP box-containing proteins have no effect. Furthermore, we identify PIP degron-containing peptides from several substrates of CRL4Cdt2 as efficient inhibitors of Pol η foci formation. By site-directed mutagenesis we show that inhibition depends on a conserved threonine residue that confers high affinity for PCNA-binding. Altogether these findings reveal an important regulative role for the CRL4Cdt2 pathway in the switch of PCNA partners on DNA damage. PMID:24423875

PIP degron proteins, substrates of CRL4Cdt2, and not PIP boxes, interfere with DNA polymerase η and κ focus formation on UV damage.

PubMed

Tsanov, Nikolay; Kermi, Chames; Coulombe, Philippe; Van der Laan, Siem; Hodroj, Dana; Maiorano, Domenico

2014-04-01

Proliferating cell nuclear antigen (PCNA) is a well-known scaffold for many DNA replication and repair proteins, but how the switch between partners is regulated is currently unclear. Interaction with PCNA occurs via a domain known as a PCNA-Interacting Protein motif (PIP box). More recently, an additional specialized PIP box has been described, the « PIP degron », that targets PCNA-interacting proteins for proteasomal degradation via the E3 ubiquitin ligase CRL4(Cdt2). Here we provide evidence that CRL4(Cdt2)-dependent degradation of PIP degron proteins plays a role in the switch of PCNA partners during the DNA damage response by facilitating accumulation of translesion synthesis DNA polymerases into nuclear foci. We show that expression of a nondegradable PIP degron (Cdt1) impairs both Pol η and Pol κ focus formation on ultraviolet irradiation and reduces cell viability, while canonical PIP box-containing proteins have no effect. Furthermore, we identify PIP degron-containing peptides from several substrates of CRL4(Cdt2) as efficient inhibitors of Pol η foci formation. By site-directed mutagenesis we show that inhibition depends on a conserved threonine residue that confers high affinity for PCNA-binding. Altogether these findings reveal an important regulative role for the CRL4(Cdt2) pathway in the switch of PCNA partners on DNA damage.

Regulation of oxidative DNA damage repair by DNA polymerase λ and MutYH by cross-talk of phosphorylation and ubiquitination

PubMed Central

Markkanen, Enni; van Loon, Barbara; Ferrari, Elena; Parsons, Jason L.; Dianov, Grigory L.; Hübscher, Ulrich

2012-01-01

It is of pivotal importance for genome stability that repair DNA polymerases (Pols), such as Pols λ and β, which all exhibit considerably reduced fidelity when replicating undamaged DNA, are tightly regulated, because their misregulation could lead to mutagenesis. Recently, we found that the correct repair of the abundant and highly miscoding oxidative DNA lesion 7,8-dihydro-8-oxo-2′-deoxyguanine (8-oxo-G) is performed by an accurate repair pathway that is coordinated by the MutY glycosylase homologue (MutYH) and Pol λ in vitro and in vivo. Pol λ is phosphorylated by Cdk2/cyclinA in late S and G2 phases of the cell cycle, promoting Pol λ stability by preventing it from being targeted for proteasomal degradation by ubiquitination. However, it has remained a mystery how the levels of Pol λ are controlled, how phosphorylation promotes its stability, and how the engagement of Pol λ in active repair complexes is coordinated. Here, we show that the E3 ligase Mule mediates the degradation of Pol λ and that the control of Pol λ levels by Mule has functional consequences for the ability of mammalian cells to deal with 8-oxo-G lesions. Furthermore, we demonstrate that phosphorylation of Pol λ by Cdk2/cyclinA counteracts its Mule-mediated degradation by promoting recruitment of Pol λ to chromatin into active 8-oxo-G repair complexes through an increase in Pol λ’s affinity to chromatin-bound MutYH. Finally, MutYH appears to promote the stability of Pol λ by binding it to chromatin. In contrast, Pol λ not engaged in active repair on chromatin is subject for proteasomal degradation. PMID:22203964

Solution Dependence of the Collisional Activation of Ubiquitin [M+7H]7+ Ions

PubMed Central

Shi, Huilin; Atlasevich, Natalya; Merenbloom, Samuel I.; Clemmer, David E.

2014-01-01

The solution dependence of gas-phase unfolding for ubiquitin [M+7H]7+ ions has been studied by ion mobility spectrometry-mass spectrometry (IMS-MS). Different acidic water:methanol solutions are used to favor the native (N), more helical (A), or unfolded (U) solution states of ubiquitin. Unfolding of gas-phase ubiquitin ions is achieved by collisional heating and newly formed structures are examined by IMS. With an activation voltage of 100 V, a selected distribution of compact structures unfolds, forming three resolvable elongated states (E1-E3). The relative populations of these elongated structures depend strongly on the solution composition. Activation of compact ions from aqueous solutions known to favor N-state ubiquitin produces mostly the E1 type elongated state, whereas, activation of compact ions from methanol containing solutions that populate A-state ubiquitin favors the E3 elongated state. Presumably, this difference arises because of differences in precursor ion structures emerging from solution. Thus, it appears that information about solution populations can be retained after ionization, selection, and activation to produce the elongated states. These data as well as others are discussed. PMID:24658799

Human CRL4DDB2 ubiquitin ligase preferentially regulates post-repair chromatin restoration of H3K56Ac through recruitment of histone chaperon CAF-1

PubMed Central

Zhu, Qianzheng; Wei, Shengcai; Sharma, Nidhi; Wani, Gulzar; He, Jinshan; Wani, Altaf A.

2017-01-01

Acetylated histone H3 lysine 56 (H3K56Ac) diminishes in response to DNA damage but is restored following DNA repair. Here, we report that CRL4DDB2 ubiquitin ligase preferentially regulates post-repair chromatin restoration of H3K56Ac through recruitment of histone chaperon CAF-1. We show that H3K56Ac accumulates at DNA damage sites. The restoration of H3K56Ac but not H3K27Ac, H3K18Ac and H3K14Ac depends on CAF-1 function, whereas all these acetylations are mediated by CBP/p300. The CRL4DDB2 components, DDB1, DDB2 and CUL4A, are also required for maintaining the H3K56Ac and H3K9Ac level in chromatin, and for restoring H3K56Ac following induction of DNA photolesions and strand breaks. Depletion of CUL4A decreases the recruitment of CAF-1 p60 and p150 to ultraviolet radiation- and phleomycin-induced DNA damage. Neddylation inhibition renders CRL4DDB2 inactive, decreases H3K56Ac level, diminishes CAF-1 recruitment and prevents H3K56Ac restoration. Mutation in the PIP box of DDB2 compromises its capability to elevate the H3K56Ac level but does not affect XPC ubiquitination. These results demonstrated a function of CRL4DDB2 in differential regulation of histone acetylation in response to DNA damage, suggesting a novel role of CRL4DDB2 in repair-driven chromatin assembly. PMID:29262658

DNA damage and repair after high LET radiation

NASA Astrophysics Data System (ADS)

O'Neill, Peter; Cucinotta, Francis; Anderson, Jennifer

Predictions from biophysical models of interactions of radiation tracks with cellular DNA indicate that clustered DNA damage sites, defined as two or more lesions formed within one or two helical turns of the DNA by passage of a single radiation track, are formed in mammalian cells. These complex DNA damage sites are regarded as a signature of ionizing radiation exposure particularly as the likelihood of clustered damage sites arising endogenously is low. For instance, it was predicted from biophysical modelling that 30-40% of low LET-induced double strand breaks (DSB), a form of clustered damage, are complex with the yield increasing to >90% for high LET radiation, consistent with the reduced reparability of DSB with increasing ionization density of the radiation. The question arises whether the increased biological effects such as mutagenesis, carcinogenesis and lethality is in part related to DNA damage complexity and/or spatial distribution of the damage sites, which may lead to small DNA fragments. With particle radiation it is also important to consider not only delta-rays which may cause clustered damaged sites and may be highly mutagenic but the non-random spatial distribution of DSB which may lead to deletions. In this overview I will concentrate on the molecular aspects of the variation of the complexity of DNA damage on radiation quality and the challenges this complexity presents the DNA damage repair pathways. I will draw on data from micro-irradiations which indicate that the repair of DSBs by non-homologous end joining is highly regulated with pathway choice and kinetics of repair dependent on the chemical complexity of the DSB. In summary the aim is to emphasis the link between the spatial distribution of energy deposition events related to the track, the molecular products formed and the consequence of damage complexity contributing to biological effects and to present some of the outstanding molecular challenges with particle radiation.

DNA damage in blood cells exposed to low-level lasers.

PubMed

Sergio, Luiz Philippe da Silva; Silva, Ana Paula Almeida da; Amorim, Philipi Freitas; Campos, Vera Maria Araújo; Magalhães, Luis Alexandre Gonçalves; de Paoli, Flavia; de Souza da Fonseca, Adenilson

2015-04-01

In regenerative medicine, there are increasing applications of low-level lasers in therapeutic protocols for treatment of diseases in soft and in bone tissues. However, there are doubts about effects on DNA, and an adequate dosimetry could improve the safety of clinical applications of these lasers. This work aimed to evaluate DNA damage in peripheral blood cells of Wistar rats induced by low-level red and infrared lasers at different fluences, powers, and emission modes according to therapeutic protocols. Peripheral blood samples were exposed to lasers and DNA damage was accessed by comet assay. In other experiments, DNA damage was accessed in blood cells by modified comet assay using formamidopyrimidine DNA glycosylase (Fpg) and endonuclease III enzymes. Data show that exposure to low-level red and infrared lasers induce DNA damage depending on fluence, power and emission mode, which are targeted by Fpg and endonuclease III. Oxidative DNA damage should be considered for therapeutic efficacy and patient safety in clinical applications based on low-level red and infrared lasers. © 2015 Wiley Periodicals, Inc.

Characterization of UVC-induced DNA damage in bloodstains: forensic implications.

PubMed

Hall, Ashley; Ballantyne, Jack

2004-09-01

The ability to detect DNA polymorphisms using molecular genetic techniques has revolutionized the forensic analysis of biological evidence. DNA typing now plays a critical role within the criminal justice system, but one of the limiting factors with the technology is that DNA isolated from biological stains recovered from the crime scene is sometimes so damaged as to be intractable to analysis. Potential remedies for damaged DNA are likely to be dependent upon the precise nature of the DNA damage present in any particular sample but, unfortunately, current knowledge of the biochemical nature, and the extent, of such DNA damage in dried biological stains is rudimentary. As a model for DNA damage assessment in biological stains recovered from crime scenes, we have subjected human bloodstains and naked DNA in the hydrated and dehydrated states to varying doses of UVC radiation. It was possible to damage the DNA sufficiently in a bloodstain to cause a standard autosomal short tandem repeat (STR) profile to be lost. However, a detailed analysis of the process, based upon assays developed to detect bipyrimidine photoproducts (BPPPs), single- and double-strand breaks, and DNA-DNA crosslinks, produced some unexpected findings. Contrary to the situation with living tissues or cells in culture, the predominant UVC-induced damage to DNA in bloodstains appears not to be pyrimidine dimers. Although some evidence for the presence of BPPPs and DNA crosslinks was obtained, the major form of UVC damage causing genetic profile loss appeared to be single-strand breaks. It was not possible, however, to preclude the possibility that a combination of damage types was responsible for the profile loss observed. We demonstrate here that a significant measure of protection against UVC-mediated genetic profile loss in dried biological stain material is afforded by the dehydrated state of the DNA and, to a lesser extent, the DNA cellular milieu.

Mobile phone radiation induces mode-dependent DNA damage in a mouse spermatocyte-derived cell line: a protective role of melatonin.

PubMed

Liu, Chuan; Gao, Peng; Xu, Shang-Cheng; Wang, Yuan; Chen, Chun-Hai; He, Min-Di; Yu, Zheng-Ping; Zhang, Lei; Zhou, Zhou

2013-11-01

To evaluate whether exposure to mobile phone radiation (MPR) can induce DNA damage in male germ cells. A mouse spermatocyte-derived GC-2 cell line was exposed to a commercial mobile phone handset once every 20 min in standby, listen, dialed or dialing modes for 24 h. DNA damage was determined using an alkaline comet assay. The levels of DNA damage were significantly increased following exposure to MPR in the listen, dialed and dialing modes. Moreover, there were significantly higher increases in the dialed and dialing modes than in the listen mode. Interestingly, these results were consistent with the radiation intensities of these modes. However, the DNA damage effects of MPR in the dialing mode were efficiently attenuated by melatonin pretreatment. These results regarding mode-dependent DNA damage have important implications for the safety of inappropriate mobile phone use by males of reproductive age and also suggest a simple preventive measure: Keeping mobile phones as far away from our body as possible, not only during conversations but during 'dialed' and 'dialing' operation modes. Since the 'dialed' mode is actually part of the standby mode, mobile phones should be kept at a safe distance from our body even during standby operation. Furthermore, the protective role of melatonin suggests that it may be a promising pharmacological candidate for preventing mobile phone use-related reproductive impairments.

A systematic analysis of factors localized to damaged chromatin reveals PARP-dependent recruitment of transcription factors

PubMed Central

Izhar, Lior; Adamson, Britt; Ciccia, Alberto; Lewis, Jedd; Pontano-Vaites, Laura; Leng, Yumei; Liang, Anthony C.; Westbrook, Thomas F.; Harper, J. Wade; Elledge, Stephen J.

2015-01-01

Localization to sites of DNA damage is a hallmark of DNA damage response (DDR) proteins. To identify new DDR factors, we screened epitope-tagged proteins for localization to sites of chromatin damaged by UV laser microirradiation and found >120 proteins that localize to damaged chromatin. These include the BAF tumor suppressor complex and the ALS candidate protein TAF15. TAF15 contains multiple domains that bind damaged chromatin in a PARP-dependent manner, suggesting a possible role as glue that tethers multiple PAR chains together. Many positives were transcription factors and >70% of randomly tested transcription factors localized to sites of DNA damage and approximately 90% were PARP-dependent for localization. Mutational analyses showed that localization to damaged chromatin is DNA-binding domain-dependent. By examining Hoechst staining patterns at damage sites, we see evidence of chromatin decompaction that is PARP-dependent. We propose that PARP-regulated chromatin remodeling at sites of damage allows transient accessibility of DNA-binding proteins. PMID:26004182

Cell-fate determination by ubiquitin-dependent regulation of translation.

PubMed

Werner, Achim; Iwasaki, Shintaro; McGourty, Colleen A; Medina-Ruiz, Sofia; Teerikorpi, Nia; Fedrigo, Indro; Ingolia, Nicholas T; Rape, Michael

2015-09-24

Metazoan development depends on the accurate execution of differentiation programs that allow pluripotent stem cells to adopt specific fates. Differentiation requires changes to chromatin architecture and transcriptional networks, yet whether other regulatory events support cell-fate determination is less well understood. Here we identify the ubiquitin ligase CUL3 in complex with its vertebrate-specific substrate adaptor KBTBD8 (CUL3(KBTBD8)) as an essential regulator of human and Xenopus tropicalis neural crest specification. CUL3(KBTBD8) monoubiquitylates NOLC1 and its paralogue TCOF1, the mutation of which underlies the neurocristopathy Treacher Collins syndrome. Ubiquitylation drives formation of a TCOF1-NOLC1 platform that connects RNA polymerase I with ribosome modification enzymes and remodels the translational program of differentiating cells in favour of neural crest specification. We conclude that ubiquitin-dependent regulation of translation is an important feature of cell-fate determination.

Parkin mediates the ubiquitination of VPS35 and modulates retromer-dependent endosomal sorting.

PubMed

Williams, Erin T; Glauser, Liliane; Tsika, Elpida; Jiang, Haisong; Islam, Shariful; Moore, Darren J

2018-06-11

Mutations in a number of genes cause familial forms of Parkinson's disease (PD), including mutations in the vacuolar protein sorting 35 ortholog (VPS35) and parkin genes. In this study, we identify a novel functional interaction between parkin and VPS35. We demonstrate that parkin interacts with and robustly ubiquitinates VPS35 in human neural cells. Familial parkin mutations are impaired in their ability to ubiquitinate VPS35. Parkin mediates the attachment of an atypical poly-ubiquitin chain to VPS35 with three lysine residues identified within the C-terminal region of VPS35 that are covalently modified by ubiquitin. Notably, parkin-mediated VPS35 ubiquitination does not promote the proteasomal degradation of VPS35. Furthermore, parkin does not influence the steady-state levels or turnover of VPS35 in neural cells and VPS35 levels are normal in the brains of parkin knockout mice. These data suggest that ubiquitination of VPS35 by parkin may instead serve a non-degradative cellular function potentially by regulating retromer-dependent sorting. Accordingly, we find that components of the retromer-associated WASH complex are markedly decreased in the brain of parkin knockout mice, suggesting that parkin may modulate WASH complex-dependent retromer sorting. Parkin gene silencing in primary cortical neurons selectively disrupts the vesicular sorting of the autophagy receptor ATG9A, a WASH-dependent retromer cargo. Parkin is not required for dopaminergic neurodegeneration induced by the expression of PD-linked D620N VPS35 in mice, consistent with VPS35 being located downstream of parkin function. Our data reveal a novel functional interaction of parkin with VPS35 that may be important for retromer-mediated endosomal sorting and PD.

Regulation of the DNA damage response by DNA-PKcs inhibitory phosphorylation of ATM

PubMed Central

Zhou, Yi; Lee, Ji-Hoon; Jiang, Wenxia; Crowe, Jennie L; Zha, Shan; Paull, Tanya T.

2017-01-01

SUMMARY Ataxia-Telangiectasia Mutated (ATM) regulates the DNA damage response as well as DNA double-strand break repair through homologous recombination. Here we show that ATM is hyperactive when the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is chemically inhibited or when the DNA-PKcs gene is deleted in human cells. Pre-incubation of ATM protein with active DNA-PKcs also significantly reduces ATM activity in vitro. We characterize several phosphorylation sites in ATM that are targets of DNA-PKcs and show that phospho-mimetic mutations at these residues significantly inhibit ATM activity and impair ATM signaling upon DNA damage. In contrast, phospho-blocking mutations at one cluster of sites increase the frequency of apoptosis during normal cell growth. DNA-PKcs, which is integral to the non-homologous end joining pathway, thus negatively regulates ATM activity through phosphorylation of ATM. These observations illuminate an important regulatory mechanism for ATM that also controls DNA repair pathway choice. PMID:27939942

Regulation of the DNA Damage Response by DNA-PKcs Inhibitory Phosphorylation of ATM.

PubMed

Zhou, Yi; Lee, Ji-Hoon; Jiang, Wenxia; Crowe, Jennie L; Zha, Shan; Paull, Tanya T

2017-01-05

Ataxia-telangiectasia mutated (ATM) regulates the DNA damage response as well as DNA double-strand break repair through homologous recombination. Here we show that ATM is hyperactive when the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is chemically inhibited or when the DNA-PKcs gene is deleted in human cells. Pre-incubation of ATM protein with active DNA-PKcs also significantly reduces ATM activity in vitro. We characterize several phosphorylation sites in ATM that are targets of DNA-PKcs and show that phospho-mimetic mutations at these residues significantly inhibit ATM activity and impair ATM signaling upon DNA damage. In contrast, phospho-blocking mutations at one cluster of sites increase the frequency of apoptosis during normal cell growth. DNA-PKcs, which is integral to the non-homologous end joining pathway, thus negatively regulates ATM activity through phosphorylation of ATM. These observations illuminate an important regulatory mechanism for ATM that also controls DNA repair pathway choice. Copyright © 2017 Elsevier Inc. All rights reserved.

Nuclear insulin-like growth factor 1 receptor phosphorylates proliferating cell nuclear antigen and rescues stalled replication forks after DNA damage.

PubMed

Waraky, Ahmed; Lin, Yingbo; Warsito, Dudi; Haglund, Felix; Aleem, Eiman; Larsson, Olle

2017-11-03

We have previously shown that the insulin-like growth factor 1 receptor (IGF-1R) translocates to the cell nucleus, where it binds to enhancer-like regions and increases gene transcription. Further studies have demonstrated that nuclear IGF-1R (nIGF-1R) physically and functionally interacts with some nuclear proteins, i.e. the lymphoid enhancer-binding factor 1 (Lef1), histone H3, and Brahma-related gene-1 proteins. In this study, we identified the proliferating cell nuclear antigen (PCNA) as a nIGF-1R-binding partner. PCNA is a pivotal component of the replication fork machinery and a main regulator of the DNA damage tolerance (DDT) pathway. We found that IGF-1R interacts with and phosphorylates PCNA in human embryonic stem cells and other cell lines. In vitro MS analysis of PCNA co-incubated with the IGF-1R kinase indicated tyrosine residues 60, 133, and 250 in PCNA as IGF-1R targets, and PCNA phosphorylation was followed by mono- and polyubiquitination. Co-immunoprecipitation experiments suggested that these ubiquitination events may be mediated by DDT-dependent E2/E3 ligases ( e.g. RAD18 and SHPRH/HLTF). Absence of IGF-1R or mutation of Tyr-60, Tyr-133, or Tyr-250 in PCNA abrogated its ubiquitination. Unlike in cells expressing IGF-1R, externally induced DNA damage in IGF-1R-negative cells caused G 1 cell cycle arrest and S phase fork stalling. Taken together, our results suggest a role of IGF-1R in DDT. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Types and Consequences of DNA Damage

EPA Science Inventory

This review provides a concise overview of the types of DNA damage and the molecular mechanisms by which a cell senses DNA damage, repairs the damage, converts the damage into a mutation, or dies as a consequence of unrepaired DNA damage. Such information is important in consid...

Lack of a p21waf1/cip -dependent G1/S checkpoint in neural stem and progenitor cells after DNA damage in vivo.

PubMed

Roque, Telma; Haton, Céline; Etienne, Olivier; Chicheportiche, Alexandra; Rousseau, Laure; Martin, Ludovic; Mouthon, Marc-André; Boussin, François D

2012-03-01

The cyclin-dependent kinase inhibitor p21(waf1/cip) mediates the p53-dependent G1/S checkpoint, which is generally considered to be a critical requirement to maintain genomic stability after DNA damage. We used staggered 5-ethynyl-2'deoxyuridine/5-bromo-2'-deoxyuridine double-labeling in vivo to investigate the cell cycle progression and the role of p21(waf1/cip) in the DNA damage response of neural stem and progenitor cells (NSPCs) after exposure of the developing mouse cortex to ionizing radiation. We observed a radiation-induced p21-dependent apoptotic response in migrating postmitotic cortical cells. However, neural stem and progenitor cells (NSPCs) did not initiate a p21(waf1/cip1) -dependent G1/S block and continued to enter S-phase at a similar rate to the non-irradiated controls. The G1/S checkpoint is not involved in the mechanisms underlying the faithful transmission of the NSPC genome and/or the elimination of critically damaged cells. These processes typically involve intra-S and G2/M checkpoints that are rapidly activated after irradiation. p21 is normally repressed in neural cells during brain development except at the G1 to G0 transition. Lack of activation of a G1/S checkpoint and apoptosis of postmitotic migrating cells after DNA damage appear to depend on the expression of p21 in neural cells, since substantial cell-to-cell variations are found in the irradiated cortex. This suggests that repression of p21 during brain development prevents the induction of the G1/S checkpoint after DNA damage. Copyright © 2011 AlphaMed Press.

The Aurora-B-dependent NoCut checkpoint prevents damage of anaphase bridges after DNA replication stress.

PubMed

Amaral, Nuno; Vendrell, Alexandre; Funaya, Charlotta; Idrissi, Fatima-Zahra; Maier, Michael; Kumar, Arun; Neurohr, Gabriel; Colomina, Neus; Torres-Rosell, Jordi; Geli, María-Isabel; Mendoza, Manuel

2016-05-01

Anaphase chromatin bridges can lead to chromosome breakage if not properly resolved before completion of cytokinesis. The NoCut checkpoint, which depends on Aurora B at the spindle midzone, delays abscission in response to chromosome segregation defects in yeast and animal cells. How chromatin bridges are detected, and whether abscission inhibition prevents their damage, remain key unresolved questions. We find that bridges induced by DNA replication stress and by condensation or decatenation defects, but not dicentric chromosomes, delay abscission in a NoCut-dependent manner. Decatenation and condensation defects lead to spindle stabilization during cytokinesis, allowing bridge detection by Aurora B. NoCut does not prevent DNA damage following condensin or topoisomerase II inactivation; however, it protects anaphase bridges and promotes cellular viability after replication stress. Therefore, the molecular origin of chromatin bridges is critical for activation of NoCut, which plays a key role in the maintenance of genome stability after replicative stress.

Method for assaying clustered DNA damages

DOEpatents

Sutherland, Betsy M.

2004-09-07

Disclosed is a method for detecting and quantifying clustered damages in DNA. In this method, a first aliquot of the DNA to be tested for clustered damages with one or more lesion-specific cleaving reagents under conditions appropriate for cleavage of the DNA to produce single-strand nicks in the DNA at sites of damage lesions. The number average molecular length (Ln) of double stranded DNA is then quantitatively determined for the treated DNA. The number average molecular length (Ln) of double stranded DNA is also quantitatively determined for a second, untreated aliquot of the DNA. The frequency of clustered damages (.PHI..sub.c) in the DNA is then calculated.

Mapping DNA damage-dependent genetic interactions in yeast via party mating and barcode fusion genetics.

PubMed

Díaz-Mejía, J Javier; Celaj, Albi; Mellor, Joseph C; Coté, Atina; Balint, Attila; Ho, Brandon; Bansal, Pritpal; Shaeri, Fatemeh; Gebbia, Marinella; Weile, Jochen; Verby, Marta; Karkhanina, Anna; Zhang, YiFan; Wong, Cassandra; Rich, Justin; Prendergast, D'Arcy; Gupta, Gaurav; Öztürk, Sedide; Durocher, Daniel; Brown, Grant W; Roth, Frederick P

2018-05-28

Condition-dependent genetic interactions can reveal functional relationships between genes that are not evident under standard culture conditions. State-of-the-art yeast genetic interaction mapping, which relies on robotic manipulation of arrays of double-mutant strains, does not scale readily to multi-condition studies. Here, we describe barcode fusion genetics to map genetic interactions (BFG-GI), by which double-mutant strains generated via en masse "party" mating can also be monitored en masse for growth to detect genetic interactions. By using site-specific recombination to fuse two DNA barcodes, each representing a specific gene deletion, BFG-GI enables multiplexed quantitative tracking of double mutants via next-generation sequencing. We applied BFG-GI to a matrix of DNA repair genes under nine different conditions, including methyl methanesulfonate (MMS), 4-nitroquinoline 1-oxide (4NQO), bleomycin, zeocin, and three other DNA-damaging environments. BFG-GI recapitulated known genetic interactions and yielded new condition-dependent genetic interactions. We validated and further explored a subnetwork of condition-dependent genetic interactions involving MAG1 , SLX4, and genes encoding the Shu complex, and inferred that loss of the Shu complex leads to an increase in the activation of the checkpoint protein kinase Rad53. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

Implications of caspase-dependent proteolytic cleavage of cyclin A1 in DNA damage-induced cell death

DOE Office of Scientific and Technical Information (OSTI.GOV)

Woo, Sang Hyeok; Seo, Sung-Keum; An, Sungkwan

Highlights: • Caspase-1 mediates doxorubicin-induced downregulation of cyclin A1. • Active caspase-1 effectively cleaved cyclin A1 at D165. • Cyclin A1 expression is involved in DNA damage-induced cell death. - Abstract: Cyclin A1 is an A-type cyclin that directly binds to CDK2 to regulate cell-cycle progression. In the present study, we found that doxorubicin decreased the expression of cyclin A1 at the protein level in A549 lung cancer cells, while markedly downregulating its mRNA levels. Interestingly, doxorubicin upregulated caspase-1 in a concentration-dependent manner, and z-YAVD-fmk, a specific inhibitor of caspase-1, reversed the doxorubicin-induced decrease in cyclin A1 in A549 lungmore » cancer and MCF7 breast cancer cells. Active caspase-1 effectively cleaved cyclin A1 at D165 into two fragments, which in vitro cleavage assays showed were further cleaved by caspase-3. Finally, we found that overexpression of cyclin A1 significantly reduced the cytotoxicity of doxorubicin, and knockdown of cyclin A1 by RNA interference enhanced the sensitivity of cells to ionizing radiation. Our data suggest a new mechanism for the downregulation of cyclin A1 by DNA-damaging stimuli that could be intimately involved in the cell death induced by DNA damage-inducing stimuli, including doxorubicin and ionizing radiation.« less

Protein Interactions in T7 DNA Replisome Facilitate DNA Damage Bypass.

PubMed

Zou, Zhenyu; Chen, Ze; Xue, Qizhen; Xu, Ying; Xiong, Jingyuan; Yang, Ping; Le, Shuai; Zhang, Huidong

2018-06-14

DNA replisome inevitably encounters DNA damage during DNA replication. T7 DNA replisome contains DNA polymerase (gp5), the processivity factor thioredoxin (trx), helicase-primase (gp4), and ssDNA binding protein (gp2.5). T7 protein interactions mediate this DNA replication. However, whether the protein interactions could promote DNA damage bypass is still little addressed. In this study, we investigated the strand-displacement DNA synthesis past 8-oxoG or O6-MeG at the synthetic DNA fork by T7 DNA replisome. DNA damage does not obviously affect the binding affinities among helicase, polymerase, and DNA fork. Relative to unmodified G, both 8-oxoG and O6-MeG, as well as GC-rich template sequence clusters, inhibit the strand-displacement DNA synthesis and produce partial extension products. Relative to gp4 ΔC-tail, gp4 promotes the DNA damage bypass. The presence of gp2.5 further promotes this bypass. Thus, the interactions of polymerase with helicase and ssDNA binidng protein faciliate the DNA damage bypass. Similarly, accessory proteins in other complicated DNA replisomes also facilitate the DNA damage bypass. This work provides the novel mechanism information of DNA damage bypass by DNA replisome. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Epigenetic Telomere Protection by Drosophila DNA Damage Response Pathways

PubMed Central

Oikemus, Sarah R; Queiroz-Machado, Joana; Lai, KuanJu; McGinnis, Nadine; Sunkel, Claudio; Brodsky, Michael H

2006-01-01

Analysis of terminal deletion chromosomes indicates that a sequence-independent mechanism regulates protection of Drosophila telomeres. Mutations in Drosophila DNA damage response genes such as atm/tefu, mre11, or rad50 disrupt telomere protection and localization of the telomere-associated proteins HP1 and HOAP, suggesting that recognition of chromosome ends contributes to telomere protection. However, the partial telomere protection phenotype of these mutations limits the ability to test if they act in the epigenetic telomere protection mechanism. We examined the roles of the Drosophila atm and atr-atrip DNA damage response pathways and the nbs homolog in DNA damage responses and telomere protection. As in other organisms, the atm and atr-atrip pathways act in parallel to promote telomere protection. Cells lacking both pathways exhibit severe defects in telomere protection and fail to localize the protection protein HOAP to telomeres. Drosophila nbs is required for both atm- and atr-dependent DNA damage responses and acts in these pathways during DNA repair. The telomere fusion phenotype of nbs is consistent with defects in each of these activities. Cells defective in both the atm and atr pathways were used to examine if DNA damage response pathways regulate telomere protection without affecting telomere specific sequences. In these cells, chromosome fusion sites retain telomere-specific sequences, demonstrating that loss of these sequences is not responsible for loss of protection. Furthermore, terminally deleted chromosomes also fuse in these cells, directly implicating DNA damage response pathways in the epigenetic protection of telomeres. We propose that recognition of chromosome ends and recruitment of HP1 and HOAP by DNA damage response proteins is essential for the epigenetic protection of Drosophila telomeres. Given the conserved roles of DNA damage response proteins in telomere function, related mechanisms may act at the telomeres of other organisms

Epigenetic telomere protection by Drosophila DNA damage response pathways.

PubMed

Oikemus, Sarah R; Queiroz-Machado, Joana; Lai, KuanJu; McGinnis, Nadine; Sunkel, Claudio; Brodsky, Michael H

2006-05-01

Analysis of terminal deletion chromosomes indicates that a sequence-independent mechanism regulates protection of Drosophila telomeres. Mutations in Drosophila DNA damage response genes such as atm/tefu, mre11, or rad50 disrupt telomere protection and localization of the telomere-associated proteins HP1 and HOAP, suggesting that recognition of chromosome ends contributes to telomere protection. However, the partial telomere protection phenotype of these mutations limits the ability to test if they act in the epigenetic telomere protection mechanism. We examined the roles of the Drosophila atm and atr-atrip DNA damage response pathways and the nbs homolog in DNA damage responses and telomere protection. As in other organisms, the atm and atr-atrip pathways act in parallel to promote telomere protection. Cells lacking both pathways exhibit severe defects in telomere protection and fail to localize the protection protein HOAP to telomeres. Drosophila nbs is required for both atm- and atr-dependent DNA damage responses and acts in these pathways during DNA repair. The telomere fusion phenotype of nbs is consistent with defects in each of these activities. Cells defective in both the atm and atr pathways were used to examine if DNA damage response pathways regulate telomere protection without affecting telomere specific sequences. In these cells, chromosome fusion sites retain telomere-specific sequences, demonstrating that loss of these sequences is not responsible for loss of protection. Furthermore, terminally deleted chromosomes also fuse in these cells, directly implicating DNA damage response pathways in the epigenetic protection of telomeres. We propose that recognition of chromosome ends and recruitment of HP1 and HOAP by DNA damage response proteins is essential for the epigenetic protection of Drosophila telomeres. Given the conserved roles of DNA damage response proteins in telomere function, related mechanisms may act at the telomeres of other organisms.

A Systematic Analysis of Factors Localized to Damaged Chromatin Reveals PARP-Dependent Recruitment of Transcription Factors.

PubMed

Izhar, Lior; Adamson, Britt; Ciccia, Alberto; Lewis, Jedd; Pontano-Vaites, Laura; Leng, Yumei; Liang, Anthony C; Westbrook, Thomas F; Harper, J Wade; Elledge, Stephen J

2015-06-09

Localization to sites of DNA damage is a hallmark of DNA damage response (DDR) proteins. To identify DDR factors, we screened epitope-tagged proteins for localization to sites of chromatin damaged by UV laser microirradiation and found >120 proteins that localize to damaged chromatin. These include the BAF tumor suppressor complex and the amyotrophic lateral sclerosis (ALS) candidate protein TAF15. TAF15 contains multiple domains that bind damaged chromatin in a poly-(ADP-ribose) polymerase (PARP)-dependent manner, suggesting a possible role as glue that tethers multiple PAR chains together. Many positives were transcription factors; > 70% of randomly tested transcription factors localized to sites of DNA damage, and of these, ∼90% were PARP dependent for localization. Mutational analyses showed that localization to damaged chromatin is DNA-binding-domain dependent. By examining Hoechst staining patterns at damage sites, we see evidence of chromatin decompaction that is PARP dependent. We propose that PARP-regulated chromatin remodeling at sites of damage allows transient accessibility of DNA-binding proteins. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Structure of a Novel DNA-binding Domain of Helicase-like Transcription Factor (HLTF) and Its Functional Implication in DNA Damage Tolerance.

PubMed

Hishiki, Asami; Hara, Kodai; Ikegaya, Yuzu; Yokoyama, Hideshi; Shimizu, Toshiyuki; Sato, Mamoru; Hashimoto, Hiroshi

2015-05-22

HLTF (helicase-like transcription factor) is a yeast RAD5 homolog found in mammals. HLTF has E3 ubiquitin ligase and DNA helicase activities, and plays a pivotal role in the template-switching pathway of DNA damage tolerance. HLTF has an N-terminal domain that has been designated the HIRAN (HIP116 and RAD5 N-terminal) domain. The HIRAN domain has been hypothesized to play a role in DNA binding; however, the structural basis of, and functional evidence for, the HIRAN domain in DNA binding has remained unclear. Here we show for the first time the crystal structure of the HIRAN domain of human HLTF in complex with DNA. The HIRAN domain is composed of six β-strands and two α-helices, forming an OB-fold structure frequently found in ssDNA-binding proteins, including in replication factor A (RPA). Interestingly, this study reveals that the HIRAN domain interacts with not only with a single-stranded DNA but also with a duplex DNA. Furthermore, the structure unexpectedly clarifies that the HIRAN domain specifically recognizes the 3'-end of DNA. These results suggest that the HIRAN domain functions as a sensor to the 3'-end of the primer strand at the stalled replication fork and that the domain facilitates fork regression. HLTF is recruited to a damaged site through the HIRAN domain at the stalled replication fork. Furthermore, our results have implications for the mechanism of template switching. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

RAD18 Activates the G2/M Checkpoint through DNA Damage Signaling to Maintain Genome Integrity after Ionizing Radiation Exposure

PubMed Central

Sasatani, Megumi; Xu, Yanbin; Kawai, Hidehiko; Cao, Lili; Tateishi, Satoshi; Shimura, Tsutomu; Li, Jianxiang; Iizuka, Daisuke; Noda, Asao; Hamasaki, Kanya; Kusunoki, Yoichiro; Kamiya, Kenji

2015-01-01

The ubiquitin ligase RAD18 is involved in post replication repair pathways via its recruitment to stalled replication forks, and its role in the ubiquitylation of proliferating cell nuclear antigen (PCNA). Recently, it has been reported that RAD18 is also recruited to DNA double strand break (DSB) sites, where it plays novel functions in the DNA damage response induced by ionizing radiation (IR). This new role is independent of PCNA ubiquitylation, but little is known about how RAD18 functions after IR exposure. Here, we describe a role for RAD18 in the IR-induced DNA damage signaling pathway at G2/M phase in the cell cycle. Depleting cells of RAD18 reduced the recruitment of the DNA damage signaling factors ATM, γH2AX, and 53BP1 to foci in cells at the G2/M phase after IR exposure, and attenuated activation of the G2/M checkpoint. Furthermore, depletion of RAD18 increased micronuclei formation and cell death following IR exposure, both in vitro and in vivo. Our data suggest that RAD18 can function as a mediator for DNA damage response signals to activate the G2/M checkpoint in order to maintain genome integrity and cell survival after IR exposure. PMID:25675240

FANCL ubiquitinates β-catenin and enhances its nuclear function

PubMed Central

Rotelli, Michael D.; Petersen, Curtis L.; Kaech, Stefanie; Nelson, Whitney D.; Yates, Jane E.; Hanlon Newell, Amy E.; Olson, Susan B.; Druker, Brian J.; Bagby, Grover C.

2012-01-01

Bone marrow failure is a nearly universal complication of Fanconi anemia. The proteins encoded by FANC genes are involved in DNA damage responses through the formation of a multisubunit nuclear complex that facilitates the E3 ubiquitin ligase activity of FANCL. However, it is not known whether loss of E3 ubiquitin ligase activity accounts for the hematopoietic stem cell defects characteristic of Fanconi anemia. Here we provide evidence that FANCL increases the activity and expression of β-catenin, a key pluripotency factor in hematopoietic stem cells. We show that FANCL ubiquitinates β-catenin with atypical ubiquitin chain extension known to have nonproteolytic functions. Specifically, β-catenin modified with lysine-11 ubiquitin chain extension efficiently activates a lymphocyte enhancer-binding factor-T cell factor reporter. We also show that FANCL-deficient cells display diminished capacity to activate β-catenin leading to reduced transcription of Wnt-responsive targets c-Myc and Cyclin D1. Suppression of FANCL expression in normal human CD34+ stem and progenitor cells results in fewer β-catenin active cells and inhibits expansion of multilineage progenitors. Together, these results suggest that diminished Wnt/β-catenin signaling may be an underlying molecular defect in FANCL-deficient hematopoietic stem cells leading to their accelerated loss. PMID:22653977

FANCL ubiquitinates β-catenin and enhances its nuclear function.

PubMed

Dao, Kim-Hien T; Rotelli, Michael D; Petersen, Curtis L; Kaech, Stefanie; Nelson, Whitney D; Yates, Jane E; Hanlon Newell, Amy E; Olson, Susan B; Druker, Brian J; Bagby, Grover C

2012-07-12

Bone marrow failure is a nearly universal complication of Fanconi anemia. The proteins encoded by FANC genes are involved in DNA damage responses through the formation of a multisubunit nuclear complex that facilitates the E3 ubiquitin ligase activity of FANCL. However, it is not known whether loss of E3 ubiquitin ligase activity accounts for the hematopoietic stem cell defects characteristic of Fanconi anemia. Here we provide evidence that FANCL increases the activity and expression of β-catenin, a key pluripotency factor in hematopoietic stem cells. We show that FANCL ubiquitinates β-catenin with atypical ubiquitin chain extension known to have nonproteolytic functions. Specifically, β-catenin modified with lysine-11 ubiquitin chain extension efficiently activates a lymphocyte enhancer-binding factor-T cell factor reporter. We also show that FANCL-deficient cells display diminished capacity to activate β-catenin leading to reduced transcription of Wnt-responsive targets c-Myc and Cyclin D1. Suppression of FANCL expression in normal human CD34(+) stem and progenitor cells results in fewer β-catenin active cells and inhibits expansion of multilineage progenitors. Together, these results suggest that diminished Wnt/β-catenin signaling may be an underlying molecular defect in FANCL-deficient hematopoietic stem cells leading to their accelerated loss.

E3 ligase Rad18 promotes monoubiquitination rather than ubiquitin chain formation by E2 enzyme Rad6

PubMed Central

Hibbert, Richard G.; Huang, Anding; Boelens, Rolf; Sixma, Titia K.

2011-01-01

In ubiquitin conjugation, different combinations of E2 and E3 enzymes catalyse either monoubiquitination or ubiquitin chain formation. The E2/E3 complex Rad6/Rad18 exclusively monoubiquitinates the proliferating cell nuclear antigen (PCNA) to signal for “error prone” DNA damage tolerance, whereas a different set of conjugation enzymes is required for ubiquitin chain formation on PCNA. Here we show that human E2 enzyme Rad6b is intrinsically capable of catalyzing ubiquitin chain formation. This activity is prevented during PCNA ubiquitination by the interaction of Rad6 with E3 enzyme Rad18. Using NMR and X-ray crystallography we show that the R6BD of Rad18 inhibits this activity by competing with ubiquitin for a noncovalent “backside” binding site on Rad6. Our findings provide mechanistic insights into how E3 enzymes can regulate the ubiquitin conjugation process. PMID:21422291

Apigenin induces DNA damage through the PKCδ-dependent activation of ATM and H2AX causing down-regulation of genes involved in cell cycle control and DNA repair

PubMed Central

Arango, Daniel; Parihar, Arti; Villamena, Frederick A.; Wang, Liwen; Freitas, Michael A.; Grotewold, Erich; Doseff, Andrea I.

2014-01-01

Apigenin, an abundant plant flavonoid, exhibits anti-proliferative and anti-carcinogenic activities through mechanisms yet not fully defined. In the present study, we show that the treatment of leukemia cells with apigenin resulted in the induction of DNA damage preceding the activation of the apoptotic program. Apigenin-induced DNA damage was mediated by p38 and protein kinase C-delta (PKCδ), yet was independent of reactive oxygen species or caspase activity. Treatment of monocytic leukemia cells with apigenin induced the phosphorylation of the ataxia-telangiectasia mutated (ATM) kinase and histone H2AX, two key regulators of the DNA damage response, without affecting the ataxia-telangiectasia mutated and Rad-3-related (ATR) kinase. Silencing and pharmacological inhibition of PKCδ abrogated ATM and H2AX phosphorylation, whereas inhibition of p38 reduced H2AX phosphorylation independently of ATM. We established that apigenin delayed cell cycle progression at G1/S and increased the number of apoptotic cells. In addition, genome-wide mRNA analyses showed that apigenin-induced DNA damage led to down-regulation of genes involved in cell-cycle control and DNA repair. Taken together, the present results show that the PKCδ-dependent activation of ATM and H2AX define the signaling networks responsible for the regulation of DNA damage promoting genome-wide mRNA alterations that result in cell cycle arrest, hence contributing to the anti-carcinogenic activities of this flavonoid. PMID:22985621

Aflatoxin B₁-Induced Developmental and DNA Damage in Caenorhabditis elegans.

PubMed

Feng, Wei-Hong; Xue, Kathy S; Tang, Lili; Williams, Phillip L; Wang, Jia-Sheng

2016-12-26

Aflatoxin B₁ (AFB₁) is a ubiquitous mycotoxin produced by toxicogenic Aspergillus species. AFB₁ has been reported to cause serious adverse health effects, such as cancers and abnormal development and reproduction, in animals and humans. AFB₁ is also a potent genotoxic mutagen that causes DNA damage in vitro and in vivo. However, the link between DNA damage and abnormal development and reproduction is unclear. To address this issue, we examined the DNA damage, germline apoptosis, growth, and reproductive toxicity following exposure to AFB₁, using Caenorhabditis elegans as a study model. Results found that AFB₁ induced DNA damage and germline apoptosis, and significantly inhibited growth and reproduction of the nematodes in a concentration-dependent manner. Exposure to AFB₁ inhibited growth or reproduction more potently in the DNA repair-deficient xpa-1 nematodes than the wild-type N2 strain. According to the relative expression level of pathway-related genes measured by real-time PCR, the DNA damage response (DDR) pathway was found to be associated with AFB₁-induced germline apoptosis, which further played an essential role in the dysfunction of growth and reproduction in C. elegans .

Regulation of the yeast RAD2 gene: DNA damage-dependent induction correlates with protein binding to regulatory sequences and their deletion influences survival.

PubMed

Siede, W; Friedberg, E C

1992-03-01

In the yeast Saccharomyces cerevisiae the RAD2 gene is absolutely required for damage-specific incision of DNA during nucleotide excision repair and is inducible by DNA-damaging agents. In the present study we correlated sensitivity to killing by DNA-damaging agents with the deletion of previously defined specific promoter elements. Deletion of the element DRE2 increased the UV sensitivity of cells in both the G1/early S and S/G2 phases of the cell cycle as well as in stationary phase. On the other hand, increased UV sensitivity associated with deletion of the sequence-related element DRE1 was restricted to cells irradiated in G1/S. Specific binding of protein(s) to the promoter elements DRE1 and DRE2 was observed under non-inducing conditions using gel retardation assays. Exposure of cells to DNA-damaging agents resulted in increased protein binding that was dependent on de novo protein synthesis.

Age-dependent oxidative stress-induced DNA damage in Down's lymphocytes.

PubMed

Zana, Marianna; Szécsényi, Anita; Czibula, Agnes; Bjelik, Annamária; Juhász, Anna; Rimanóczy, Agnes; Szabó, Krisztina; Vetró, Agnes; Szucs, Péter; Várkonyi, Agnes; Pákáski, Magdolna; Boda, Krisztina; Raskó, István; Janka, Zoltán; Kálmán, János

2006-06-30

The aim of the present study was to investigate the oxidative status of lymphocytes from children (n=7) and adults (n=18) with Down's syndrome (DS). The basal oxidative condition, the vulnerability to in vitro hydrogen peroxide exposure, and the repair capacity were measured by means of the damage-specific alkaline comet assay. Significantly and age-independently elevated numbers of single strand breaks and oxidized bases (pyrimidines and purines) were found in the nuclear DNA of the lymphocytes in the DS group in the basal condition. These results may support the role of an increased level of endogenous oxidative stress in DS and are similar to those previously demonstrated in Alzheimer's disease. In the in vitro oxidative stress-induced state, a markedly higher extent of DNA damage was observed in DS children as compared with age- and gender-matched healthy controls, suggesting that young trisomic lymphocytes are more sensitive to oxidative stress than normal ones. However, the repair ability itself was not found to be deteriorated in either DS children or DS adults.

Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65

PubMed Central

Kazlauskaite, Agne; Kondapalli, Chandana; Gourlay, Robert; Campbell, David G.; Ritorto, Maria Stella; Hofmann, Kay; Alessi, Dario R.; Knebel, Axel; Trost, Matthias; Muqit, Miratul M. K.

2014-01-01

We have previously reported that the Parkinson's disease-associated kinase PINK1 (PTEN-induced putative kinase 1) is activated by mitochondrial depolarization and stimulates the Parkin E3 ligase by phosphorylating Ser65 within its Ubl (ubiquitin-like) domain. Using phosphoproteomic analysis, we identified a novel ubiquitin phosphopeptide phosphorylated at Ser65 that was enriched 14-fold in HEK (human embryonic kidney)-293 cells overexpressing wild-type PINK1 stimulated with the mitochondrial uncoupling agent CCCP (carbonyl cyanide m-chlorophenylhydrazone), to activate PINK1, compared with cells expressing kinase-inactive PINK1. Ser65 in ubiquitin lies in a similar motif to Ser65 in the Ubl domain of Parkin. Remarkably, PINK1 directly phosphorylates Ser65 of ubiquitin in vitro. We undertook a series of experiments that provide striking evidence that Ser65-phosphorylated ubiquitin (ubiquitinPhospho−Ser65) functions as a critical activator of Parkin. First, we demonstrate that a fragment of Parkin lacking the Ubl domain encompassing Ser65 (ΔUbl-Parkin) is robustly activated by ubiquitinPhospho−Ser65, but not by non-phosphorylated ubiquitin. Secondly, we find that the isolated Parkin Ubl domain phosphorylated at Ser65 (UblPhospho−Ser65) can also activate ΔUbl-Parkin similarly to ubiquitinPhospho−Ser65. Thirdly, we establish that ubiquitinPhospho−Ser65, but not non-phosphorylated ubiquitin or UblPhospho−Ser65, activates full-length wild-type Parkin as well as the non-phosphorylatable S65A Parkin mutant. Fourthly, we provide evidence that optimal activation of full-length Parkin E3 ligase is dependent on PINK1-mediated phosphorylation of both Parkin at Ser65 and ubiquitin at Ser65, since only mutation of both proteins at Ser65 completely abolishes Parkin activation. In conclusion, the findings of the present study reveal that PINK1 controls Parkin E3 ligase activity not only by phosphorylating Parkin at Ser65, but also by phosphorylating ubiquitin at Ser65

Quantitative Proteomics Reveals Dynamic Interactions of the Minichromosome Maintenance Complex (MCM) in the Cellular Response to Etoposide Induced DNA Damage.

PubMed

Drissi, Romain; Dubois, Marie-Line; Douziech, Mélanie; Boisvert, François-Michel

2015-07-01

The minichromosome maintenance complex (MCM) proteins are required for processive DNA replication and are a target of S-phase checkpoints. The eukaryotic MCM complex consists of six proteins (MCM2-7) that form a heterohexameric ring with DNA helicase activity, which is loaded on chromatin to form the pre-replication complex. Upon entry in S phase, the helicase is activated and opens the DNA duplex to recruit DNA polymerases at the replication fork. The MCM complex thus plays a crucial role during DNA replication, but recent work suggests that MCM proteins could also be involved in DNA repair. Here, we employed a combination of stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics with immunoprecipitation of green fluorescent protein-tagged fusion proteins to identify proteins interacting with the MCM complex, and quantify changes in interactions in response to DNA damage. Interestingly, the MCM complex showed very dynamic changes in interaction with proteins such as Importin7, the histone chaperone ASF1, and the Chromodomain helicase DNA binding protein 3 (CHD3) following DNA damage. These changes in interactions were accompanied by an increase in phosphorylation and ubiquitination on specific sites on the MCM proteins and an increase in the co-localization of the MCM complex with γ-H2AX, confirming the recruitment of these proteins to sites of DNA damage. In summary, our data indicate that the MCM proteins is involved in chromatin remodeling in response to DNA damage. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Leucocytes DNA damage in mice exposed to JS-118 by the comet assay.

PubMed

Zhang, Tao; Hu, Jiye; Zhang, Yuchao; Zhao, Qianfei; Ning, Jun

2011-09-01

JS-118 is an extensively used insecticide in China. The present study investigated the genotoxic effect of JS-118 on whole blood at 24, 48, 72 and 96 h by using alkaline comet assay. Male Kunming mice were given 6.25, 12.5, 25, 50 and 100 mg/kg BW of JS-118 intraperitoneally. A statistically significant increase in all comet parameters indicating DNA damage was observed at 24 h post-treatment (p < 0.05). A clear concentration-dependent increase of DNA damage was revealed as evident by the OTM (arbitrary units), tail length (µm) and tail DNA (%). From 48 h post-treatment, a gradual decrease in mean comet parameters was noted. By 96 h of post-treatment, the mean comet tail length reached control levels indicating repair of damaged DNA. This study on mice showed different DNA damage depending on the concentration of JS-118 and the period of treatment. The present study provided further information of the potential risk of the genetic damage caused by JS-118.

The DNA damage response during mitosis.

PubMed

Heijink, Anne Margriet; Krajewska, Małgorzata; van Vugt, Marcel A T M

2013-10-01

Cells are equipped with a cell-intrinsic signaling network called the DNA damage response (DDR). This signaling network recognizes DNA lesions and initiates various downstream pathways to coordinate a cell cycle arrest with the repair of the damaged DNA. Alternatively, the DDR can mediate clearance of affected cells that are beyond repair through apoptosis or senescence. The DDR can be activated in response to DNA damage throughout the cell cycle, although the extent of DDR signaling is different in each cell cycle phase. Especially in response to DNA double strand breaks, only a very marginal response was observed during mitosis. Early on it was recognized that cells which are irradiated during mitosis continued division without repairing broken chromosomes. Although these initial observations indicated diminished DNA repair and lack of an acute DNA damage-induced cell cycle arrest, insight into the mechanistic re-wiring of DDR signaling during mitosis was only recently provided. Different mechanisms appear to be at play to inactivate specific signaling axes of the DDR network in mitosis. Importantly, mitotic cells not simply inactivate the entire DDR, but appear to mark their DNA damage for repair after mitotic exit. Since the treatment of cancer frequently involves agents that induce DNA damage as well as agents that block mitotic progression, it is clinically relevant to obtain a better understanding of how cancer cells deal with DNA damage during interphase versus mitosis. In this review, the molecular details concerning DDR signaling during mitosis as well as the consequences of encountering DNA damage during mitosis for cellular fate are discussed. Copyright © 2013 Elsevier B.V. All rights reserved.

XRN2 Links Transcription Termination to DNA Damage and Replication Stress

PubMed Central

Patidar, Praveen L.; Motea, Edward A.; Dang, Tuyen T.; Manley, James L.

2016-01-01

XRN2 is a 5’-3’ exoribonuclease implicated in transcription termination. Here we demonstrate an unexpected role for XRN2 in the DNA damage response involving resolution of R-loop structures and prevention of DNA double-strand breaks (DSBs). We show that XRN2 undergoes DNA damage-inducible nuclear re-localization, co-localizing with 53BP1 and R loops, in a transcription and R-loop-dependent process. XRN2 loss leads to increased R loops, genomic instability, replication stress, DSBs and hypersensitivity of cells to various DNA damaging agents. We demonstrate that the DSBs that arise with XRN2 loss occur at transcriptional pause sites. XRN2-deficient cells also exhibited an R-loop- and transcription-dependent delay in DSB repair after ionizing radiation, suggesting a novel role for XRN2 in R-loop resolution, suppression of replication stress, and maintenance of genomic stability. Our study highlights the importance of regulating transcription-related activities as a critical component in maintaining genetic stability. PMID:27437695

XRN2 Links Transcription Termination to DNA Damage and Replication Stress.

PubMed

Morales, Julio C; Richard, Patricia; Patidar, Praveen L; Motea, Edward A; Dang, Tuyen T; Manley, James L; Boothman, David A

2016-07-01

XRN2 is a 5'-3' exoribonuclease implicated in transcription termination. Here we demonstrate an unexpected role for XRN2 in the DNA damage response involving resolution of R-loop structures and prevention of DNA double-strand breaks (DSBs). We show that XRN2 undergoes DNA damage-inducible nuclear re-localization, co-localizing with 53BP1 and R loops, in a transcription and R-loop-dependent process. XRN2 loss leads to increased R loops, genomic instability, replication stress, DSBs and hypersensitivity of cells to various DNA damaging agents. We demonstrate that the DSBs that arise with XRN2 loss occur at transcriptional pause sites. XRN2-deficient cells also exhibited an R-loop- and transcription-dependent delay in DSB repair after ionizing radiation, suggesting a novel role for XRN2 in R-loop resolution, suppression of replication stress, and maintenance of genomic stability. Our study highlights the importance of regulating transcription-related activities as a critical component in maintaining genetic stability.

Quantitative Profiling of DNA Damage and Apoptotic Pathways in UV Damaged Cells Using PTMScan Direct

PubMed Central

Stokes, Matthew P.; Silva, Jeffrey C.; Jia, Xiaoying; Lee, Kimberly A.; Polakiewicz, Roberto D.; Comb, Michael J.

2013-01-01

Traditional methods for analysis of peptides using liquid chromatography and tandem mass spectrometry (LC-MS/MS) lack the specificity to comprehensively monitor specific biological processes due to the inherent duty cycle limitations of the MS instrument and the stochastic nature of the analytical platform. PTMScan Direct is a novel, antibody-based method that allows quantitative LC-MS/MS profiling of specific peptides from proteins that reside in the same signaling pathway. New PTMScan Direct reagents have been produced that target peptides from proteins involved in DNA Damage/Cell Cycle and Apoptosis/Autophagy pathways. Together, the reagents provide access to 438 sites on 237 proteins in these signaling cascades. These reagents have been used to profile the response to UV damage of DNA in human cell lines. UV damage was shown to activate canonical DNA damage response pathways through ATM/ATR-dependent signaling, stress response pathways and induce the initiation of apoptosis, as assessed by an increase in the abundance of peptides corresponding to cleaved, activated caspases. These data demonstrate the utility of PTMScan Direct as a multiplexed assay for profiling specific cellular responses to various stimuli, such as UV damage of DNA. PMID:23344034

Alpha-phellandrene-induced DNA damage and affect DNA repair protein expression in WEHI-3 murine leukemia cells in vitro.

PubMed

Lin, Jen-Jyh; Wu, Chih-Chung; Hsu, Shu-Chun; Weng, Shu-Wen; Ma, Yi-Shih; Huang, Yi-Ping; Lin, Jaung-Geng; Chung, Jing-Gung

2015-11-01

Although there are few reports regarding α-phellandrene (α-PA), a natural compound from Schinus molle L. essential oil, there is no report to show that α-PA induced DNA damage and affected DNA repair associated protein expression. Herein, we investigated the effects of α-PA on DNA damage and repair associated protein expression in murine leukemia cells. Flow cytometric assay was used to measure the effects of α-PA on total cell viability and the results indicated that α-PA induced cell death. Comet assay and 4,6-diamidino-2-phenylindole dihydrochloride staining were used for measuring DNA damage and condensation, respectively, and the results indicated that α-PA induced DNA damage and condensation in a concentration-dependent manner. DNA gel electrophoresis was used to examine the DNA damage and the results showed that α-PA induced DNA damage in WEHI-3 cells. Western blotting assay was used to measure the changes of DNA damage and repair associated protein expression and the results indicated that α-PA increased p-p53, p-H2A.X, 14-3-3-σ, and MDC1 protein expression but inhibited the protein of p53, MGMT, DNA-PK, and BRCA-1. © 2014 Wiley Periodicals, Inc.

Mechanisms of mutagenesis: DNA replication in the presence of DNA damage

PubMed Central

Liu, Binyan; Xue, Qizhen; Tang, Yong; Cao, Jia; Guengerich, F. Peter; Zhang, Huidong

2017-01-01

Environmental mutagens cause DNA damage that disturbs replication and produces mutations, leading to cancer and other diseases. We discuss mechanisms of mutagenesis resulting from DNA damage, from the level of DNA replication by a single polymerase to the complex DNA replisome of some typical model organisms (including bacteriophage T7, T4, Sulfolobus solfataricus, E. coli, yeast and human). For a single DNA polymerase, DNA damage can affect replication in three major ways: reducing replication fidelity, causing frameshift mutations, and blocking replication. For the DNA replisome, protein interactions and the functions of accessory proteins can yield rather different results even with a single DNA polymerase. The mechanism of mutation during replication performed by the DNA replisome is a long-standing question. Using new methods and techniques, the replisomes of certain organisms and human cell extracts can now be investigated with regard to the bypass of DNA damage. In this review, we consider the molecular mechanism of mutagenesis resulting from DNA damage in replication at the levels of single DNA polymerases and complex DNA replisomes, including translesion DNA synthesis. PMID:27234563

RING-type ubiquitin ligase McCPN1 catalyzes UBC8-dependent protein ubiquitination and interacts with Argonaute 4 in halophyte ice plant.

PubMed

Li, Chang-Hua; Chiang, Chih-Pin; Yang, Jun-Yi; Ma, Chia-Jou; Chen, Yu-Chan; Yen, Hungchen Emilie

2014-07-01

RING-type copines are a small family of plant-specific RING-type ubiquitin ligases. They contain an N-terminal myristoylation site for membrane anchoring, a central copine domain for substrate recognition, and a C-terminal RING domain for E2 docking. RING-type copine McCPN1 (copine1) from halophyte ice plant (Mesembryanthemum crystallinum L.) was previously identified from a salt-induced cDNA library. In this work, we characterize the activity, expression, and localization of McCPN1 in ice plant. An in vitro ubiquitination assay of McCPN1 was performed using two ice plant UBCs, McUBC1 and McUBC2, characterized from the same salt-induced cDNA library. The results showed that McUBC2, a member of the UBC8 family, stimulated the autoubiquitination activity of McCPN1, while McUBC1, a homolog of the UBC35 family, did not. The results indicate that McCPN1 has selective E2-dependent E3 ligase activity. We found that McCPN1 localizes primarily on the plasma membrane and in the nucleus of plant cells. Under salt stress, the accumulation of McCPN1 in the roots increases. A yeast two-hybrid screen was used to search for potential McCPN1-interacting partners using a library constructed from salt-stressed ice plants. Screening with full-length McCPN1 identified several independent clones containing partial Argonaute 4 (AGO4) sequence. Subsequent agro-infiltration, protoplast two-hybrid analysis, and bimolecular fluorescence complementation assay confirmed that McCPN1 and AGO4 interacted in vivo in the nucleus of plant cells. The possible involvement of a catalyzed degradation of AGO4 by McCPN1 in response to salt stress is discussed. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Immunohistochemical detection of ubiquitin-positive intracytoplasmic eosinophilic inclusion bodies in diffuse alveolar damage.

PubMed

Yamada, T; Uehara, K; Kawanishi, R; Mizutani, T; Sunagawa, K; Araya, J; Kawabata, Y

2006-06-01

To clarify the relationship between ubiquitin-positive pneumocytes and intracytoplasmic eosinophilic inclusion bodies (IB) in patients who died of diffuse alveolar damage (DAD). Eighteen patients with DAD were studied, in whom hyaline membranes were present in one or more out of five sections from each lobe of the lungs and 15 patients with no DAD. Light microscopy revealed hyaline membrane in over 25% of lobes from 18 patients with DAD. The cytoplasm of pneumocytes from six of 18 cases of DAD contained IB. Immunohistochemically, all IBs were characteristically positive for both ubiquitin (Ub) and cytokeratin KL-1. Cytoplasmic granules were also Ub+ in four cases of DAD without IB. IB+ or Ub+ pneumocytes were undetectable in non-DAD patients. We evaluated DAD severity based on hyaline membrane formation; the mean score in DAD with IB (3.60; n = 6) was significantly higher than that in Ub- (2.92; n = 8). Ub+ pneumocytes were found with or without IB among those cases with high DAD scores. These findings suggest that disordered proteolysis in the Ub-mediated proteasome system leads to the accumulation of abnormal ubiquitinated protein, which includes cytokeratin, in pneumocytes. This is the first report to suggest that Ub+ pneumocytes are associated with disease severity in patients with DAD.

Ubiquitin-dependent endocytosis, trafficking and turnover of neuronal membrane proteins

PubMed Central

Schwarz, Lindsay A.; Patrick, Gentry N.

2011-01-01

Extracellular signaling between cells is often transduced via receptors that reside at the cell membrane. In neurons this receptor-mediated signaling can promote a variety of cellular events such as differentiation, axon outgrowth and guidance, synaptic development and function. Endocytic membrane trafficking of receptors can ensure that the strength and duration of an extracellular signal is properly regulated. The covalent modification of membrane proteins by ubiquitin is a key biological mechanism to control receptor internalization and endocytic sorting to recycling and degradative pathways in many cell types. In this review we highlight recent findings regarding the ubiquitin-dependent trafficking and turnover of receptors in neurons and the implications for neuronal development and function. PMID:21884797

Effect of ionizing radiation exposure on Trypanosoma cruzi ubiquitin-proteasome system.

PubMed

Cerqueira, Paula G; Passos-Silva, Danielle G; Vieira-da-Rocha, João P; Mendes, Isabela Cecilia; de Oliveira, Karla A; Oliveira, Camila F B; Vilela, Liza F F; Nagem, Ronaldo A P; Cardoso, Joseane; Nardelli, Sheila C; Krieger, Marco A; Franco, Glória R; Macedo, Andrea M; Pena, Sérgio D J; Schenkman, Sérgio; Gomes, Dawidson A; Guerra-Sá, Renata; Machado, Carlos R

2017-03-01

In recent years, proteasome involvement in the damage response induced by ionizing radiation (IR) became evident. However, whether proteasome plays a direct or indirect role in IR-induced damage response still unclear. Trypanosoma cruzi is a human parasite capable of remarkable high tolerance to IR, suggesting a highly efficient damage response system. Here, we investigate the role of T. cruzi proteasome in the damage response induced by IR. We exposed epimastigotes to high doses of gamma ray and we analyzed the expression and subcellular localization of several components of the ubiquitin-proteasome system. We show that proteasome inhibition increases IR-induced cell growth arrest and proteasome-mediated proteolysis is altered after parasite exposure. We observed nuclear accumulation of 19S and 20S proteasome subunits in response to IR treatments. Intriguingly, the dynamic of 19S particle nuclear accumulation was more similar to the dynamic observed for Rad51 nuclear translocation than the observed for 20S. In the other hand, 20S increase and nuclear translocation could be related with an increase of its regulator PA26 and high levels of proteasome-mediated proteolysis in vitro. The intersection between the opposed peaks of 19S and 20S protein levels was marked by nuclear accumulation of both 20S and 19S together with Ubiquitin, suggesting a role of ubiquitin-proteasome system in the nuclear protein turnover at the time. Our results revealed the importance of proteasome-mediated proteolysis in T. cruzi IR-induced damage response suggesting that proteasome is also involved in T. cruzi IR tolerance. Moreover, our data support the possible direct/signaling role of 19S in DNA damage repair. Based on these results, we speculate that spatial and temporal differences between the 19S particle and 20S proteasome controls proteasome multiple roles in IR damage response. Copyright © 2017 Elsevier B.V. All rights reserved.

A defined role for multiple Fanconi anemia gene products in DNA-damage-associated ubiquitination.

PubMed

Tan, Winnie; Deans, Andrew J

2017-06-01

Fanconi anemia (FA) is an inherited blood disorder that causes bone marrow failure and high predisposition to cancers. The FA pathway guards the cell's genome stability by orchestrating the repair of interstrand cross-linking during the S phase of the cell cycle, preventing the chromosomal instability that is a key event in bone marrow failure syndrome. Central to the FA pathway is loss of monoubiquitinated forms of the Fanconi proteins FANCI and FANCD2, a process that is normally mediated by a "core complex" of seven other Fanconi proteins. Each protein, when mutated, can cause FA. The FA core-complex-catalyzed reaction is critical for signaling DNA cross-link damage such as that induced by chemotherapies. Here, we present a perspective on the current understanding of FANCI and FANCD2 monoubiquitination-mediated DNA repair. Our recent biochemical reconstitution of the monoubiquitination (and deubiquitination) reactions creates a paradigm for understanding FA. Further biochemical analysis will create new opportunities to address the leukemic phenotype of FA patients. Copyright © 2017 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

Mechanisms of mutagenesis: DNA replication in the presence of DNA damage.

PubMed

Liu, Binyan; Xue, Qizhen; Tang, Yong; Cao, Jia; Guengerich, F Peter; Zhang, Huidong

2016-01-01

Environmental mutagens cause DNA damage that disturbs replication and produces mutations, leading to cancer and other diseases. We discuss mechanisms of mutagenesis resulting from DNA damage, from the level of DNA replication by a single polymerase to the complex DNA replisome of some typical model organisms (including bacteriophage T7, T4, Sulfolobus solfataricus, Escherichia coli, yeast and human). For a single DNA polymerase, DNA damage can affect replication in three major ways: reducing replication fidelity, causing frameshift mutations, and blocking replication. For the DNA replisome, protein interactions and the functions of accessory proteins can yield rather different results even with a single DNA polymerase. The mechanism of mutation during replication performed by the DNA replisome is a long-standing question. Using new methods and techniques, the replisomes of certain organisms and human cell extracts can now be investigated with regard to the bypass of DNA damage. In this review, we consider the molecular mechanism of mutagenesis resulting from DNA damage in replication at the levels of single DNA polymerases and complex DNA replisomes, including translesion DNA synthesis. Copyright © 2016 Elsevier B.V. All rights reserved.

RAD9-dependent G1 arrest defines a second checkpoint for damaged DNA in the cell cycle of Saccharomyces cerevisiae.

PubMed

Siede, W; Friedberg, A S; Friedberg, E C

1993-09-01

Exposure of the yeast Saccharomyces cerevisiae to ultraviolet (UV) light, the UV-mimetic chemical 4-nitroquinoline-1-oxide (4NQO), or gamma radiation after release from G1 arrest induced by alpha factor results in delayed resumption of the cell cycle. As is the case with G2 arrest following ionizing radiation damage [Weinert, T. A. & Hartwell, L. H. (1988) Science 241, 317-322], the normal execution of DNA damage-induced G1 arrest depends on a functional yeast RAD9 gene. We suggest that the RAD9 gene product may interact with cellular components common to the G1/S and G2/M transition points in the cell cycle of this yeast. These observations define a checkpoint in the eukaryotic cell cycle that may facilitate the repair of lesions that are otherwise processed to lethal and/or mutagenic damage during DNA replication. This checkpoint apparently operates after the mating pheromone-induced G1 arrest point but prior to replicative DNA synthesis, S phase-associated maximal induction of histone H2A mRNA, and bud emergence.

Oxidant stress in mitochondrial DNA damage, autophagy and inflammation in atherosclerosis

PubMed Central

Ding, Zufeng; Liu, Shijie; Wang, Xianwei; Khaidakov, Magomed; Dai, Yao; Mehta, Jawahar L.

2013-01-01

Our studies in HUVECs show that ox-LDL induced autophagy and damaged mtDNA leading to TLR9 expression. LOX-1 antibody or the ROS inhibitor apocynin attenuated ox-LDL-mediated autophagy, mtDNA damage and TLR9 expression, suggesting that these events are LOX-1 and ROS-dependent phenomena. Experiments using siRNA to DNase II indicated that DNase II digests mtDNA to protect the tissue from inflammation. Next, we studied and found intense autophagy, TLR9 expression and inflammatory signals (CD45 and CD68) in the aortas of LDLR knockout mice fed high cholesterol diet. Deletion of LOX-1 (LDLR/LOX-1 double knockout mice) attenuated autophagy, TLR9 expression as well as CD45 and CD68. Damaged mtDNA signal was also very high in LDLR knockout mice aortas, and this signal was attenuated by LOX-1 deletion. Thus, it appears that oxidative stress-mediated damaged mtDNA that escapes autophagy induces a potent inflammatory response in atherosclerosis. PMID:23326634

TRAF Family Member-associated NF-κB Activator (TANK) Inhibits Genotoxic Nuclear Factor κB Activation by Facilitating Deubiquitinase USP10-dependent Deubiquitination of TRAF6 Ligase*

PubMed Central

Wang, Wei; Huang, Xuan; Xin, Hong-Bo; Fu, Mingui; Xue, Aimin; Wu, Zhao-Hui

2015-01-01

DNA damage-induced NF-κB activation plays a critical role in regulating cellular response to genotoxic stress. However, the molecular mechanisms controlling the magnitude and duration of this genotoxic NF-κB signaling cascade are poorly understood. We recently demonstrated that genotoxic NF-κB activation is regulated by reversible ubiquitination of several essential mediators involved in this signaling pathway. Here we show that TRAF family member-associated NF-κB activator (TANK) negatively regulates NF-κB activation by DNA damage via inhibiting ubiquitination of TRAF6. Despite the lack of a deubiquitination enzyme domain, TANK has been shown to negatively regulate the ubiquitination of TRAF proteins. We found TANK formed a complex with MCPIP1 (also known as ZC3H12A) and a deubiquitinase, USP10, which was essential for the USP10-dependent deubiquitination of TRAF6 and the resolution of genotoxic NF-κB activation upon DNA damage. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated deletion of TANK in human cells significantly enhanced NF-κB activation by genotoxic treatment, resulting in enhanced cell survival and increased inflammatory cytokine production. Furthermore, we found that the TANK-MCPIP1-USP10 complex also decreased TRAF6 ubiquitination in cells treated with IL-1β or LPS. In accordance, depletion of USP10 enhanced NF-κB activation induced by IL-1β or LPS. Collectively, our data demonstrate that TANK serves as an important negative regulator of NF-κB signaling cascades induced by genotoxic stress and IL-1R/Toll-like receptor stimulation in a manner dependent on MCPIP1/USP10-mediated TRAF6 deubiquitination. PMID:25861989

Repair of Oxidative DNA Damage in Saccharomyces cerevisiae.

PubMed

Chalissery, Jisha; Jalal, Deena; Al-Natour, Zeina; Hassan, Ahmed H

2017-03-01

Malfunction of enzymes that detoxify reactive oxygen species leads to oxidative attack on biomolecules including DNA and consequently activates various DNA repair pathways. The nature of DNA damage and the cell cycle stage at which DNA damage occurs determine the appropriate repair pathway to rectify the damage. Oxidized DNA bases are primarily repaired by base excision repair and nucleotide incision repair. Nucleotide excision repair acts on lesions that distort DNA helix, mismatch repair on mispaired bases, and homologous recombination and non-homologous end joining on double stranded breaks. Post-replication repair that overcomes replication blocks caused by DNA damage also plays a crucial role in protecting the cell from the deleterious effects of oxidative DNA damage. Mitochondrial DNA is also prone to oxidative damage and is efficiently repaired by the cellular DNA repair machinery. In this review, we discuss the DNA repair pathways in relation to the nature of oxidative DNA damage in Saccharomyces cerevisiae. Copyright © 2017 Elsevier B.V. All rights reserved.

AtMMS21, an SMC5/6 complex subunit, is involved in stem cell niche maintenance and DNA damage responses in Arabidopsis roots.

PubMed

Xu, Panglian; Yuan, Dongke; Liu, Ming; Li, Chunxin; Liu, Yiyang; Zhang, Shengchun; Yao, Nan; Yang, Chengwei

2013-04-01

Plants maintain stem cells in meristems to sustain lifelong growth; these stem cells must have effective DNA damage responses to prevent mutations that can propagate to large parts of the plant. However, the molecular links between stem cell functions and DNA damage responses remain largely unexplored. Here, we report that the small ubiquitin-related modifier E3 ligase AtMMS21 (for methyl methanesulfonate sensitivity gene21) acts to maintain the root stem cell niche by mediating DNA damage responses in Arabidopsis (Arabidopsis thaliana). Mutation of AtMMS21 causes defects in the root stem cell niche during embryogenesis and postembryonic stages. AtMMS21 is essential for the proper expression of stem cell niche-defining transcription factors. Moreover, mms21-1 mutants are hypersensitive to DNA-damaging agents, have a constitutively increased DNA damage response, and have more DNA double-strand breaks (DSBs) in the roots. Also, mms21-1 mutants exhibit spontaneous cell death within the root stem cell niche, and treatment with DSB-inducing agents increases this cell death, suggesting that AtMMS21 is required to prevent DSB-induced stem cell death. We further show that AtMMS21 functions as a subunit of the STRUCTURAL MAINTENANCE OF CHROMOSOMES5/6 complex, an evolutionarily conserved chromosomal ATPase required for DNA repair. These data reveal that AtMMS21 acts in DSB amelioration and stem cell niche maintenance during Arabidopsis root development.

Dynamic changes to survivin subcellular localization are initiated by DNA damage

PubMed Central

Asumen, Maritess Gay; Ifeacho, Tochukwu V; Cockerham, Luke; Pfandl, Christina; Wall, Nathan R

2010-01-01

Subcellular distribution of the apoptosis inhibitor survivin and its ability to relocalize as a result of cell cycle phase or therapeutic insult has led to the hypothesis that these subcellular pools may coincide with different survivin functions. The PIK kinases (ATM, ATR and DNA-PK) phosphorylate a variety of effector substrates that propagate DNA damage signals, resulting in various biological outputs. Here we demonstrate that subcellular repartitioning of survivin in MCF-7 cells as a result of UV light-mediated DNA damage is dependent upon DNA damage-sensing proteins as treatment with the pan PIK kinase inhibitor wortmannin repartitioned survivin in the mitochondria and diminished it from the cytosol and nucleus. Mitochondrial redistribution of survivin, such as was recorded after wortmannin treatment, occurred in cells lacking any one of the three DNA damage sensing protein kinases: DNA-PK, ATM or ATR. However, failed survivin redistribution from the mitochondria in response to low-dose UV occurred only in the cells lacking ATM, implying that ATM may be the primary kinase involved in this process. Taken together, this data implicates survivian’s subcellular distribution is a dynamic physiological process that appears responsive to UV light-initiated DNA damage and that its distribution may be responsible for its multifunctionality. PMID:20856848

DNA Damage Response Genes and the Development of Cancer Metastasis

PubMed Central

Broustas, Constantinos G.; Lieberman, Howard B.

2014-01-01

DNA damage response genes play vital roles in the maintenance of a healthy genome. Defects in cell cycle checkpoint and DNA repair genes, especially mutation or aberrant downregulation, are associated with a wide spectrum of human disease, including a predisposition to the development of neurodegenerative conditions and cancer. On the other hand, upregulation of DNA damage response and repair genes can also cause cancer, as well as increase resistance of cancer cells to DNA damaging therapy. In recent years, it has become evident that many of the genes involved in DNA damage repair have additional roles in tumorigenesis, most prominently by acting as transcriptional (co-) factors. Although defects in these genes are causally connected to tumor initiation, their role in tumor progression is more controversial and it seems to depend on tumor type. In some tumors like melanoma, cell cycle checkpoint/DNA repair gene upregulation is associated with tumor metastasis, whereas in a number of other cancers the opposite has been observed. Several genes that participate in the DNA damage response, such as RAD9, PARP1, BRCA1, ATM and TP53 have been associated with metastasis by a number of in vitro biochemical and cellular assays, by examining human tumor specimens by immunohistochemistry or by DNA genomewide gene expression profiling. Many of these genes act as transcriptional effectors to regulate other genes implicated in the pathogenesis of cancer. Furthermore, they are aberrantly expressed in numerous human tumors and are causally related to tumorigenesis. However, whether the DNA damage repair function of these genes is required to promote metastasis or another activity is responsible (e.g., transcription control) has not been determined. Importantly, despite some compelling in vitro evidence, investigations are still needed to demonstrate the role of cell cycle checkpoint and DNA repair genes in regulating metastatic phenotypes in vivo. PMID:24397478

Chemotherapy inhibits skeletal muscle ubiquitin-proteasome-dependent proteolysis.

PubMed

Tilignac, Thomas; Temparis, Sandrine; Combaret, Lydie; Taillandier, Daniel; Pouch, Marie-Noëlle; Cervek, Matjaz; Cardenas, Diana M; Le Bricon, Thierry; Debiton, Eric; Samuels, Susan E; Madelmont, Jean-Claude; Attaix, Didier

2002-05-15

Chemotherapy has cachectic effects, but it is unknown whether cytostatic agents alter skeletal muscle proteolysis. We hypothesized that chemotherapy-induced alterations in protein synthesis should result in the increased incidence of abnormal proteins, which in turn should stimulate ubiquitin-proteasome-dependent proteolysis. The effects of the nitrosourea cystemustine were investigated in skeletal muscles from both healthy and colon 26 adenocarcinoma-bearing mice, an appropriate model for testing the impact of cytostatic agents. Muscle wasting was seen in both groups of mice 4 days after a single cystemustine injection, and the drug further increased the loss of muscle proteins already apparent in tumor-bearing animals. Cystemustine cured the tumor-bearing mice with 100% efficacy. Surprisingly, within 11 days of treatment, rates of muscle proteolysis progressively decreased below basal levels observed in healthy control mice and contributed to the cessation of muscle wasting. Proteasome-dependent proteolysis was inhibited by mechanisms that include reduced mRNA levels for 20S and 26S proteasome subunits, decreased protein levels of 20S proteasome subunits and the S14 non-ATPase subunit of the 26S proteasome, and impaired chymotrypsin- and trypsin-like activities of the enzyme. A combination of cisplatin and ifosfamide, two drugs that are widely used in the treatment of cancer patients, also depressed the expression of proteasomal subunits in muscles from rats bearing the MatB adenocarcinoma below basal levels. Thus, a down-regulation of ubiquitin-proteasome-dependent proteolysis is observed with various cytostatic agents and contributes to reverse the chemotherapy-induced muscle wasting.

Regulation of HTLV-1 Tax Stability, Cellular Trafficking and NF-κB Activation by the Ubiquitin-Proteasome Pathway

PubMed Central

Lavorgna, Alfonso; Harhaj, Edward William

2014-01-01

Human T-cell leukemia virus type 1 (HTLV-1) is a complex retrovirus that infects CD4+ T cells and causes adult T-cell leukemia/lymphoma (ATLL) in 3%–5% of infected individuals after a long latent period. HTLV-1 Tax is a trans-activating protein that regulates viral gene expression and also modulates cellular signaling pathways to enhance T-cell proliferation and cell survival. The Tax oncoprotein promotes T-cell transformation, in part via constitutive activation of the NF-κB transcription factor; however, the underlying mechanisms remain unknown. Ubiquitination is a type of post-translational modification that occurs in a three-step enzymatic cascade mediated by E1, E2 and E3 enzymes and regulates protein stability as well as signal transduction, protein trafficking and the DNA damage response. Emerging studies indicate that Tax hijacks the ubiquitin machinery to activate ubiquitin-dependent kinases and downstream NF-κB signaling. Tax interacts with the E2 conjugating enzyme Ubc13 and is conjugated on C-terminal lysine residues with lysine 63-linked polyubiquitin chains. Tax K63-linked polyubiquitination may serve as a platform for signaling complexes since this modification is critical for interactions with NEMO and IKK. In addition to NF-κB signaling, mono- and polyubiquitination of Tax also regulate its subcellular trafficking and stability. Here, we review recent advances in the diverse roles of ubiquitin in Tax function and how Tax usurps the ubiquitin-proteasome pathway to promote oncogenesis. PMID:25341660

Detection of damaged DNA bases by DNA glycosylase enzymes.

PubMed

Friedman, Joshua I; Stivers, James T

2010-06-22

A fundamental and shared process in all forms of life is the use of DNA glycosylase enzymes to excise rare damaged bases from genomic DNA. Without such enzymes, the highly ordered primary sequences of genes would rapidly deteriorate. Recent structural and biophysical studies are beginning to reveal a fascinating multistep mechanism for damaged base detection that begins with short-range sliding of the glycosylase along the DNA chain in a distinct conformation we call the search complex (SC). Sliding is frequently punctuated by the formation of a transient "interrogation" complex (IC) where the enzyme extrahelically inspects both normal and damaged bases in an exosite pocket that is distant from the active site. When normal bases are presented in the exosite, the IC rapidly collapses back to the SC, while a damaged base will efficiently partition forward into the active site to form the catalytically competent excision complex (EC). Here we review the unique problems associated with enzymatic detection of rare damaged DNA bases in the genome and emphasize how each complex must have specific dynamic properties that are tuned to optimize the rate and efficiency of damage site location.

Detection of Damaged DNA Bases by DNA Glycosylase Enzymes†

PubMed Central

Friedman, Joshua I.; Stivers, James T.

2010-01-01

A fundamental and shared process in all forms of life is the use of DNA glycosylase enzymes to excise rare damaged bases from genomic DNA. Without such enzymes, the highly-ordered primary sequences of genes would rapidly deteriorate. Recent structural and biophysical studies are beginning to reveal a fascinating multistep mechanism for damaged base detection that begins with short-range sliding of the glycosylase along the DNA chain in a distinct conformation we refer to as the search complex (SC). Sliding is frequently punctuated by the formation of a transient “interrogation” complex (IC) where the enzyme extrahelically inspects both normal and damaged bases in an exosite pocket that is distant from the active site. When normal bases are presented in the exosite, the IC rapidly collapses back to the SC, while a damaged base will efficiently partition forward into the active site to form the catalytically competent excision complex (EC). Here we review the unique problems associated with enzymatic detection of rare damaged DNA bases in the genome, and emphasize how each complex must have specific dynamic properties that are tuned to optimize the rate and efficiency of damage site location. PMID:20469926

Independent mechanisms recruit the cohesin loader protein NIPBL to sites of DNA damage.

PubMed

Bot, Christopher; Pfeiffer, Annika; Giordano, Fosco; Manjeera, Dharani E; Dantuma, Nico P; Ström, Lena

2017-03-15

NIPBL is required to load the cohesin complex on to DNA. While the canonical role of cohesin is to couple replicated sister chromatids together until the onset of mitosis, it also promotes tolerance to DNA damage. Here, we show that NIPBL is recruited to DNA damage throughout the cell cycle via independent mechanisms, influenced by type of damage. First, the heterochromatin protein HP1γ (also known as CBX3) recruits NIPBL to DNA double-strand breaks (DSBs) through the corresponding HP1-binding motif within the N-terminus. By contrast, the C-terminal HEAT repeat domain is unable to recruit NIPBL to DSBs but independently targets NIPBL to laser microirradiation-induced DNA damage. Each mechanism is dependent on the RNF8 and RNF168 ubiquitylation pathway, while the recruitment of the HEAT repeat domain requires further ATM or ATR activity. Thus, NIPBL has evolved a sophisticated response to damaged DNA that is influenced by the form of damage, suggesting a highly dynamic role for NIPBL in maintaining genomic stability. © 2017. Published by The Company of Biologists Ltd.

Formation and degradation of nitrogen mustard-induced MGMT-DNA crosslinking in 16HBE cells.

PubMed

Cheng, Jin; Ye, Feng; Dan, Guorong; Zhao, Yuanpeng; Zhao, Jiqing; Zou, Zhongmin

2017-08-15

N-methyl-2,2-di(chloroethyl)amine (HN2) is a kind of bifunctional alkyltating agent, which can react with nucleophilic groups in DNA and/or protein to form HN2-bridged crosslinking of target molecules, such as DNA-protein crosslinkings (DPC). O 6 -methylguanine-DNA methyltransferase (MGMT) is a DNA damage repair enzyme which solely repairs alkyl adduct on DNA directly. However, MGMT was detected to act as a protein cross-linked with DNA via alkylation in presence of HN2, and unexpectedly turned into a DNA damage enhancer in the form of MGMT-DNA cross-link (mDPC). Present study aimed to explore the possible ways to lessen the incorporation of MGMT into DPC as well as to save it for DNA repair. To find out the influencing factors of mDPC formation and cleavage, human bronchial epithelial cell line 16HBE was exposed to HN2 and the factors related with MGMT expression and degradation were investigated. When c-Myc, a negative transcriptional factor of MGMT was inhibited by 10058-F4, MGMT expression and mDPC formation were increased, and more γ-H2AX was also detected. Sustained treatment with O 6 BG, a specific exogenous substrate and depleter of MGMT, could reduce the level of MGMT and mDPC formation. In contrast, a transient 1h pre-treatment of O 6 GB before HN2 exposure would cause a high MGMT and mDPC level. MGMT was increasingly ubiquitinated after HN2 exposure in a time-dependent manner. At the same time, MGMT was also SUMOylated with a downward time-dependent manner compared to its ubiquitination. Inhibitors of E1, E2 or E3 ligases of ubiqutination all led to the accumulation of mDPC and total-DPC (tDPC) with the difference as that mDPC was sensitive to E1 inhibitor while tDPC more sensitive to E2 and E3 inhibitor. Our results demonstrated the control of mDPC level could be realized through transcription inhibitory effect of c-Myc, O 6 GB application, and the acceleration of mDPC ubiquitination and subsequent degradation. Copyright © 2017. Published by Elsevier

The Blue Light-Dependent Polyubiquitination and Degradation of Arabidopsis Cryptochrome2 Requires Multiple E3 Ubiquitin Ligases.

PubMed

Liu, Qing; Wang, Qin; Liu, Bin; Wang, Wei; Wang, Xu; Park, Joon; Yang, Zhenming; Du, Xinglin; Bian, Mingdi; Lin, Chentao

2016-10-01

Cryptochromes are blue light receptors regulated by light-dependent ubiquitination and degradation in both plant and animal lineages. The Arabidopsis genome encodes two cryptochromes, CRY1 and CRY2, of which CRY2 undergoes blue light-dependent ubiquitination and 26S proteasome-dependent degradation. The molecular mechanism regulating blue light-dependent proteolysis of CRY2 is still not fully understood. We found that the F-box proteins ZEITLUPE (ZTL) and Lov Kelch Protein2 (LKP2), which mediate blue light suppression of degradation of the CRY2 signaling partner CIB1, are not required for the blue light-dependent CRY2 degradation. We further showed that the previously reported function of the COP1-SPA1 protein complex in blue light-dependent CRY2 degradation is more likely to be attributable to its cullin 4 (CUL4)-based E3 ubiquitin ligase activity than its activity as the cryptochrome signaling partner. However, the blue light-dependent CRY2 degradation is only partially impaired in the cul4 mutant, the cop1-5 null mutant and the spa1234 quadruple mutant, suggesting a possible involvement of additional E3 ubiquitin ligases in the regulation of CRY2. Consistent with this hypothesis, we demonstrated that the blue light-dependent CRY2 degradation is significantly impaired in the temperature-sensitive cul1 mutant allele (axr6-3), especially under the non-permissive temperature. Based on these and other results presented, we propose that photoexcited CRY2 undergoes Lys48-linked polyubiquitination catalyzed by the CUL4- and CUL1-based E3 ubiquitin ligases. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Linking loss of sodium-iodide symporter expression to DNA damage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lyckesvärd, Madeleine Nordén; Department of Medical Chemistry and Cell Biology, University of Gothenburg, Göteborg; Kapoor, Nirmal

Radiotherapy of thyroid cancer with I-131 is abrogated by inherent loss of radioiodine uptake due to loss of sodium iodide symporter (NIS) expression in poorly differentiated tumor cells. It is also known that ionizing radiation per se down-regulates NIS (the stunning effect), but the mechanism is unknown. Here we investigated whether loss of NIS-mediated iodide transport may be elicited by DNA damage. Calicheamicin, a fungal toxin that specifically cleaves double-stranded DNA, induced a full scale DNA damage response mediated by the ataxia-telangiectasia mutated (ATM) kinase in quiescent normal thyrocytes. At sublethal concentrations (<1 nM) calicheamicin blocked NIS mRNA expression andmore » transepithelial iodide transport as stimulated by thyrotropin; loss of function occurred at a much faster rate than after I-131 irradiation. KU-55933, a selective ATM kinase inhibitor, partly rescued NIS expression and iodide transport in DNA-damaged cells. Prolonged ATM inhibition in healthy cells also repressed NIS-mediated iodide transport. ATM-dependent loss of iodide transport was counteracted by IGF-1. Together, these findings indicate that NIS, the major iodide transporter of the thyroid gland, is susceptible to DNA damage involving ATM-mediated mechanisms. This uncovers novel means of poor radioiodine uptake in thyroid cells subjected to extrinsic or intrinsic genotoxic stress. - Highlights: • DNA damage inhibits polarized iodide transport in normal thyroid cells. • Down-regulation of NIS expression is mediated by activation of the ATM kinase. • Long-term ATM inhibition also represses NIS-mediated iodide transport. • IGF-1 rescues NIS expression and iodide transport in DNA-damaged cells.« less

PTEN positively regulates UVB-induced DNA damage repair

PubMed Central

Ming, Mei; Feng, Li; Shea, Christopher R.; Soltani, Keyoumars; Zhao, Baozhong; Han, Weinong; Smart, Robert C.; Trempus, Carol S.; He, Yu-Ying

2011-01-01

Non-melanoma skin cancer is the most common cancer in the U.S., where DNA-damaging UVB radiation from the sun remains the major environmental risk factor. However, the critical genetic targets of UVB radiation are undefined. Here we show that attenuating PTEN in epidermal keratinocytes is a predisposing factor for UVB-induced skin carcinogenesis in mice. In skin papilloma and squamous cell carcinoma (SCC), levels of PTEN were reduced compared to skin lacking these lesions. Likewise, there was a reduction in PTEN levels in human premalignant actinic keratosis and malignant SCC, supporting a key role for PTEN in human skin cancer formation and progression. PTEN downregulation impaired the capacity of global genomic nucleotide excision repair (GG-NER), a critical mechanism for removing UVB-induced mutagenic DNA lesions. In contrast to the response to ionizing radiation, PTEN downregulation prolonged UVB-induced growth arrest and increased the activation of the Chk1 DNA damage pathway in an AKT-independent manner, likely due to reduced DNA repair. PTEN loss also suppressed expression of the key GG-NER protein xeroderma pigmentosum C (XPC) through the AKT/p38 signaling axis. Reconstitution of XPC levels in PTEN-inhibited cells restored GG-NER capacity. Taken together, our findings define PTEN as an essential genomic gatekeeper in the skin, through its ability to positively regulate XPC-dependent GG-NER following DNA damage. PMID:21771908

Phosphoramide mustard exposure induces DNA adduct formation and the DNA damage repair response in rat ovarian granulosa cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ganesan, Shanthi, E-mail: shanthig@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

Phosphoramide mustard (PM), the ovotoxic metabolite of the anti-cancer agent cyclophosphamide (CPA), destroys rapidly dividing cells by forming NOR-G-OH, NOR-G and G-NOR-G adducts with DNA, potentially leading to DNA damage. A previous study demonstrated that PM induces ovarian DNA damage in rat ovaries. To investigate whether PM induces DNA adduct formation, DNA damage and induction of the DNA repair response, rat spontaneously immortalized granulosa cells (SIGCs) were treated with vehicle control (1% DMSO) or PM (3 or 6 μM) for 24 or 48 h. Cell viability was reduced (P < 0.05) after 48 h of exposure to 3 or 6more » μM PM. The NOR-G-OH DNA adduct was detected after 24 h of 6 μM PM exposure, while the more cytotoxic G-NOR-G DNA adduct was formed after 48 h by exposure to both PM concentrations. Phosphorylated H2AX (γH2AX), a marker of DNA double stranded break occurrence, was also increased by PM exposure, coincident with DNA adduct formation. Additionally, induction of genes (Atm, Parp1, Prkdc, Xrcc6, and Brca1) and proteins (ATM, γH2AX, PARP-1, PRKDC, XRCC6, and BRCA1) involved in DNA repair were observed in both a time- and dose-dependent manner. These data support that PM induces DNA adduct formation in ovarian granulosa cells, induces DNA damage and elicits the ovarian DNA repair response. - Highlights: • PM forms ovarian DNA adducts. • DNA damage marker γH2AX increased by PM exposure. • PM induces ovarian DNA double strand break repair.« less

C/EBPα regulates CRL4Cdt2-mediated degradation of p21 in response to UVB-induced DNA damage to control the G1/S checkpoint

PubMed Central

Hall, Jonathan R; Bereman, Michael S; Nepomuceno, Angelito I; Thompson, Elizabeth A; Muddiman, David C; Smart, Robert C

2014-01-01

The bZIP transcription factor, C/EBPα is highly inducible by UVB and other DNA damaging agents in keratinocytes. C/EBPα-deficient keratinocytes fail to undergo cell cycle arrest in G1 in response to UVB-induced DNA damage and mice lacking epidermal C/EBPα are highly susceptible to UVB-induced skin cancer. The mechanism through which C/EBPα regulates the cell cycle checkpoint in response to DNA damage is unknown. Here we report untreated C/EBPα-deficient keratinocytes have normal levels of the cyclin-dependent kinase inhibitor, p21, however, UVB-treated C/EBPα-deficient keratinocytes fail to up-regulate nuclear p21 protein levels despite normal up-regulation of Cdkn1a mRNA levels. UVB-treated C/EBPα-deficient keratinocytes displayed a 4-fold decrease in nuclear p21 protein half-life due to the increased proteasomal degradation of p21 via the E3 ubiquitin ligase CRL4Cdt2. Cdt2 is the substrate recognition subunit of CRL4Cdt2 and Cdt2 mRNA and protein levels were up-regulated in UVB-treated C/EBPα-deficient keratinocytes. Knockdown of Cdt2 restored p21 protein levels in UVB-treated C/EBPα-deficient keratinocytes. Lastly, the failure to accumulate p21 in response to UVB in C/EBPα-deficient keratinocytes resulted in decreased p21 interactions with critical cell cycle regulatory proteins, increased CDK2 activity, and inappropriate entry into S-phase. These findings reveal C/EBPα regulates G1/S cell cycle arrest in response to DNA damage via the control of CRL4Cdt2 mediated degradation of p21. PMID:25483090

Small RNA-mediated repair of UV-induced DNA lesions by the DNA DAMAGE-BINDING PROTEIN 2 and ARGONAUTE 1

PubMed Central

Schalk, Catherine; Cognat, Valérie; Graindorge, Stéfanie; Vincent, Timothée; Voinnet, Olivier; Molinier, Jean

2017-01-01

As photosynthetic organisms, plants need to prevent irreversible UV-induced DNA lesions. Through an unbiased, genome-wide approach, we have uncovered a previously unrecognized interplay between Global Genome Repair and small interfering RNAs (siRNAs) in the recognition of DNA photoproducts, prevalently in intergenic regions. Genetic and biochemical approaches indicate that, upon UV irradiation, the DNA DAMAGE-BINDING PROTEIN 2 (DDB2) and ARGONAUTE 1 (AGO1) of Arabidopsis thaliana form a chromatin-bound complex together with 21-nt siRNAs, which likely facilitates recognition of DNA damages in an RNA/DNA complementary strand-specific manner. The biogenesis of photoproduct-associated siRNAs involves the noncanonical, concerted action of RNA POLYMERASE IV, RNA-DEPENDENT RNA POLYMERASE-2, and DICER-LIKE-4. Furthermore, the chromatin association/dissociation of the DDB2-AGO1 complex is under the control of siRNA abundance and DNA damage signaling. These findings reveal unexpected nuclear functions for DCL4 and AGO1, and shed light on the interplay between small RNAs and DNA repair recognition factors at damaged sites. PMID:28325872

Voltammetric Behavior of o-Nitrophenol and Damage to DNA

PubMed Central

Zhang, Da-Peng; Wu, Wei-Li; Long, Hai-Yan; Liu, Yun-Chun; Yang, Zhou-Sheng

2008-01-01

The electrochemical behavior of o-nitrophenol was studied in detail with a glassy carbon electrode (GCE). The dependence of peak potential on pH indicated that equivalent electrons and protons were involved in the process of o-nitrophenol reduction. The interaction of o-nitrophenol with calf thymus DNA was investigated by adding DNA to the o-nitrophenol solution and by immobilizing DNA on GCE, respectively. The peak current decrement and peak potential shift in presence of DNA indicated that o-nitrophenol could interact with DNA. The result was demonstrated that the in situ DNA damage was detected by differential pulse voltammetry after the o-nitrophenol was electrochemically reduced. PMID:19325751

Recent Advancements in DNA Damage-Transcription Crosstalk and High-Resolution Mapping of DNA Breaks.

PubMed

Vitelli, Valerio; Galbiati, Alessandro; Iannelli, Fabio; Pessina, Fabio; Sharma, Sheetal; d'Adda di Fagagna, Fabrizio

2017-08-31

Until recently, DNA damage arising from physiological DNA metabolism was considered a detrimental by-product for cells. However, an increasing amount of evidence has shown that DNA damage could have a positive role in transcription activation. In particular, DNA damage has been detected in transcriptional elements following different stimuli. These physiological DNA breaks are thought to be instrumental for the correct expression of genomic loci through different mechanisms. In this regard, although a plethora of methods are available to precisely map transcribed regions and transcription start sites, commonly used techniques for mapping DNA breaks lack sufficient resolution and sensitivity to draw a robust correlation between DNA damage generation and transcription. Recently, however, several methods have been developed to map DNA damage at single-nucleotide resolution, thus providing a new set of tools to correlate DNA damage and transcription. Here, we review how DNA damage can positively regulate transcription initiation, the current techniques for mapping DNA breaks at high resolution, and how these techniques can benefit future studies of DNA damage and transcription.

ATM-activated autotaxin (ATX) propagates inflammation and DNA damage in lung epithelial cells: a new mode of action for silica-induced DNA damage?

PubMed

Zheng, Huiyuan; Högberg, Johan; Stenius, Ulla

2017-12-07

Silica exposure is a common risk factor for lung cancer. It has been claimed that key elements in cancer development are activation of inflammatory cells that indirectly induce DNA damage and proliferative stimuli in respiratory epithelial cells. We studied DNA damage induced by silica particles in respiratory epithelial cells and focused the role of the signaling enzyme autotaxin (ATX). A549 and 16 bronchial epithelial cells (16HBE) lung epithelial cells were exposed to silica particles. Reactive oxygen species (ROS), NOD-like receptor family pyrin domain containing-3 (NLRP3) inflammasome activation, ATX, ataxia telangiectasia mutated (ATM), and DNA damage (γH2AX, pCHK1, pCHK2, comet assay) were end points. Low doses of silica induced NLRP3 activation, DNA damage accumulation, and ATM phosphorylation. A novel finding was that ATM induced ATX generation and secretion. Not only silica but also rotenone, camptothecin and H2O2 activated ATX via ATM, suggesting that ATX is part of a generalized ATM response to double-strand breaks (DSBs). Surprisingly, ATX inhibition mitigated DNA damage accumulation at later time points (6-16 h), and ATX transfection caused NLRP3 activation and DNA damage. Furthermore, the product of ATX enzymatic activity, lysophosphatidic acid, recapitulated the effects of ATX transfection. These data indicate an ATM-ATX-dependent loop that propagates inflammation and DSB accumulation, making low doses of silica effective inducers of DSBs in epithelial cells. We conclude that an ATM-ATX axis interconnects DSBs with silica-induced inflammation and propagates these effects in epithelial cells. Further studies of this adverse outcome pathway may give an accurate assessment of the lowest doses of silica that causes cancer. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

TRAF Family Member-associated NF-κB Activator (TANK) Inhibits Genotoxic Nuclear Factor κB Activation by Facilitating Deubiquitinase USP10-dependent Deubiquitination of TRAF6 Ligase.

PubMed

Wang, Wei; Huang, Xuan; Xin, Hong-Bo; Fu, Mingui; Xue, Aimin; Wu, Zhao-Hui

2015-05-22

DNA damage-induced NF-κB activation plays a critical role in regulating cellular response to genotoxic stress. However, the molecular mechanisms controlling the magnitude and duration of this genotoxic NF-κB signaling cascade are poorly understood. We recently demonstrated that genotoxic NF-κB activation is regulated by reversible ubiquitination of several essential mediators involved in this signaling pathway. Here we show that TRAF family member-associated NF-κB activator (TANK) negatively regulates NF-κB activation by DNA damage via inhibiting ubiquitination of TRAF6. Despite the lack of a deubiquitination enzyme domain, TANK has been shown to negatively regulate the ubiquitination of TRAF proteins. We found TANK formed a complex with MCPIP1 (also known as ZC3H12A) and a deubiquitinase, USP10, which was essential for the USP10-dependent deubiquitination of TRAF6 and the resolution of genotoxic NF-κB activation upon DNA damage. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated deletion of TANK in human cells significantly enhanced NF-κB activation by genotoxic treatment, resulting in enhanced cell survival and increased inflammatory cytokine production. Furthermore, we found that the TANK-MCPIP1-USP10 complex also decreased TRAF6 ubiquitination in cells treated with IL-1β or LPS. In accordance, depletion of USP10 enhanced NF-κB activation induced by IL-1β or LPS. Collectively, our data demonstrate that TANK serves as an important negative regulator of NF-κB signaling cascades induced by genotoxic stress and IL-1R/Toll-like receptor stimulation in a manner dependent on MCPIP1/USP10-mediated TRAF6 deubiquitination. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Treacher Collins syndrome TCOF1 protein cooperates with NBS1 in the DNA damage response.

PubMed

Ciccia, Alberto; Huang, Jen-Wei; Izhar, Lior; Sowa, Mathew E; Harper, J Wade; Elledge, Stephen J

2014-12-30

The signal transduction pathway of the DNA damage response (DDR) is activated to maintain genomic integrity following DNA damage. The DDR promotes genomic integrity by regulating a large network of cellular activities that range from DNA replication and repair to transcription, RNA splicing, and metabolism. In this study we define an interaction between the DDR factor NBS1 and TCOF1, a nucleolar protein that regulates ribosomal DNA (rDNA) transcription and is mutated in Treacher Collins syndrome. We show that NBS1 relocalizes to nucleoli after DNA damage in a manner dependent on TCOF1 and on casein kinase II and ATM, which are known to modify TCOF1 by phosphorylation. Moreover, we identify a putative ATM phosphorylation site that is required for NBS1 relocalization to nucleoli in response to DNA damage. Last, we report that TCOF1 promotes cellular resistance to DNA damaging agents. Collectively, our findings identify TCOF1 as a DDR factor that could cooperate with ATM and NBS1 to suppress inappropriate rDNA transcription and maintain genomic integrity after DNA damage.

Treacher Collins syndrome TCOF1 protein cooperates with NBS1 in the DNA damage response

PubMed Central

Ciccia, Alberto; Huang, Jen-Wei; Izhar, Lior; Sowa, Mathew E.; Harper, J. Wade; Elledge, Stephen J.

2014-01-01

The signal transduction pathway of the DNA damage response (DDR) is activated to maintain genomic integrity following DNA damage. The DDR promotes genomic integrity by regulating a large network of cellular activities that range from DNA replication and repair to transcription, RNA splicing, and metabolism. In this study we define an interaction between the DDR factor NBS1 and TCOF1, a nucleolar protein that regulates ribosomal DNA (rDNA) transcription and is mutated in Treacher Collins syndrome. We show that NBS1 relocalizes to nucleoli after DNA damage in a manner dependent on TCOF1 and on casein kinase II and ATM, which are known to modify TCOF1 by phosphorylation. Moreover, we identify a putative ATM phosphorylation site that is required for NBS1 relocalization to nucleoli in response to DNA damage. Last, we report that TCOF1 promotes cellular resistance to DNA damaging agents. Collectively, our findings identify TCOF1 as a DDR factor that could cooperate with ATM and NBS1 to suppress inappropriate rDNA transcription and maintain genomic integrity after DNA damage. PMID:25512513

DNA damage signaling regulates age-dependent proliferative capacity of quiescent inner ear supporting cells

PubMed Central

Laos, Maarja; Anttonen, Tommi; Kirjavainen, Anna; Hällström, Taija af; Laiho, Marikki; Pirvola, Ulla

2014-01-01

Supporting cells (SCs) of the cochlear (auditory) and vestibular (balance) organs hold promise as a platform for therapeutic regeneration of the sensory hair cells. Prior data have shown proliferative restrictions of adult SCs forced to re-enter the cell cycle. By comparing juvenile and adult SCs in explant cultures, we have here studied how proliferative restrictions are linked with DNA damage signaling. Cyclin D1 overexpression, used to stimulate cell cycle re-entry, triggered higher proliferative activity of juvenile SCs. Phosphorylated form of histone H2AX (γH2AX) and p53 binding protein 1 (53BP1) were induced in a foci-like pattern in SCs of both ages as an indication of DNA double-strand break formation and activated DNA damage response. Compared to juvenile SCs, γH2AX and the repair protein Rad51 were resolved with slower kinetics in adult SCs, accompanied by increased apoptosis. Consistent with the in vitro data, in a Rb mutant mouse model in vivo, cell cycle re-entry of SCs was associated with γH2AX foci induction. In contrast to cell cycle reactivation, pharmacological stimulation of SC-to-hair-cell transdifferentiation in vitro did not trigger γH2AX. Thus, DNA damage and its prolonged resolution are critical barriers in the efforts to stimulate proliferation of the adult inner ear SCs. PMID:25063730

Phosphoramide mustard exposure induces DNA adduct formation and the DNA damage repair response in rat ovarian granulosa cells.

PubMed

Ganesan, Shanthi; Keating, Aileen F

2015-02-01

Phosphoramide mustard (PM), the ovotoxic metabolite of the anti-cancer agent cyclophosphamide (CPA), destroys rapidly dividing cells by forming NOR-G-OH, NOR-G and G-NOR-G adducts with DNA, potentially leading to DNA damage. A previous study demonstrated that PM induces ovarian DNA damage in rat ovaries. To investigate whether PM induces DNA adduct formation, DNA damage and induction of the DNA repair response, rat spontaneously immortalized granulosa cells (SIGCs) were treated with vehicle control (1% DMSO) or PM (3 or 6μM) for 24 or 48h. Cell viability was reduced (P<0.05) after 48h of exposure to 3 or 6μM PM. The NOR-G-OH DNA adduct was detected after 24h of 6μM PM exposure, while the more cytotoxic G-NOR-G DNA adduct was formed after 48h by exposure to both PM concentrations. Phosphorylated H2AX (γH2AX), a marker of DNA double stranded break occurrence, was also increased by PM exposure, coincident with DNA adduct formation. Additionally, induction of genes (Atm, Parp1, Prkdc, Xrcc6, and Brca1) and proteins (ATM, γH2AX, PARP-1, PRKDC, XRCC6, and BRCA1) involved in DNA repair were observed in both a time- and dose-dependent manner. These data support that PM induces DNA adduct formation in ovarian granulosa cells, induces DNA damage and elicits the ovarian DNA repair response. Copyright © 2014 Elsevier Inc. All rights reserved.

Oxidative DNA damage background estimated by a system model of base excision repair

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sokhansanj, B A; Wilson, III, D M

Human DNA can be damaged by natural metabolism through free radical production. It has been suggested that the equilibrium between innate damage and cellular DNA repair results in an oxidative DNA damage background that potentially contributes to disease and aging. Efforts to quantitatively characterize the human oxidative DNA damage background level based on measuring 8-oxoguanine lesions as a biomarker have led to estimates varying over 3-4 orders of magnitude, depending on the method of measurement. We applied a previously developed and validated quantitative pathway model of human DNA base excision repair, integrating experimentally determined endogenous damage rates and model parametersmore » from multiple sources. Our estimates of at most 100 8-oxoguanine lesions per cell are consistent with the low end of data from biochemical and cell biology experiments, a result robust to model limitations and parameter variation. Our results show the power of quantitative system modeling to interpret composite experimental data and make biologically and physiologically relevant predictions for complex human DNA repair pathway mechanisms and capacity.« less

Link between DNA damage and centriole disengagement/reduplication in untransformed human cells.

PubMed

Douthwright, Stephen; Sluder, Greenfield

2014-10-01

The radiation and radiomimetic drugs used to treat human tumors damage DNA in both cancer cells and normal proliferating cells. Centrosome amplification after DNA damage is well established for transformed cell types but is sparsely reported and not fully understood in untransformed cells. We characterize centriole behavior after DNA damage in synchronized untransformed human cells. One hour treatment of S phase cells with the radiomimetic drug, Doxorubicin, prolongs G2 by at least 72 h, though 14% of the cells eventually go through mitosis in that time. By 72 h after DNA damage we observe a 52% incidence of centriole disengagement plus a 10% incidence of extra centrioles. We find that either APC/C or Plk activities can disengage centrioles after DNA damage, though they normally work in concert. All disengaged centrioles are associated with γ-tubulin and maturation markers and thus, should in principle be capable of reduplicating and organizing spindle poles. The low incidence of reduplication of disengaged centrioles during G2 is due to the p53-dependent expression of p21 and the consequent loss of Cdk2 activity. We find that 26% of the cells going through mitosis after DNA damage contain disengaged or extra centrioles. This could produce genomic instability through transient or persistent spindle multipolarity. Thus, for cancer patients the use of DNA damaging therapies raises the chances of genomic instability and evolution of transformed characteristics in proliferating normal cell populations. © 2014 Wiley Periodicals, Inc.

Radiation damage to DNA in DNA-protein complexes.

PubMed

Spotheim-Maurizot, M; Davídková, M

2011-06-03

The most aggressive product of water radiolysis, the hydroxyl (OH) radical, is responsible for the indirect effect of ionizing radiations on DNA in solution and aerobic conditions. According to radiolytic footprinting experiments, the resulting strand breaks and base modifications are inhomogeneously distributed along the DNA molecule irradiated free or bound to ligands (polyamines, thiols, proteins). A Monte-Carlo based model of simulation of the reaction of OH radicals with the macromolecules, called RADACK, allows calculating the relative probability of damage of each nucleotide of DNA irradiated alone or in complexes with proteins. RADACK calculations require the knowledge of the three dimensional structure of DNA and its complexes (determined by X-ray crystallography, NMR spectroscopy or molecular modeling). The confrontation of the calculated values with the results of the radiolytic footprinting experiments together with molecular modeling calculations show that: (1) the extent and location of the lesions are strongly dependent on the structure of DNA, which in turns is modulated by the base sequence and by the binding of proteins and (2) the regions in contact with the protein can be protected against the attack by the hydroxyl radicals via masking of the binding site and by scavenging of the radicals. 2011 Elsevier B.V. All rights reserved.

The organophosphate insecticide chlorpyrifos confers its genotoxic effects by inducing DNA damage and cell apoptosis.

PubMed

Li, Diqiu; Huang, Qingchun; Lu, Miaoqing; Zhang, Lei; Yang, Zhichuan; Zong, Mimi; Tao, Liming

2015-09-01

The organophosphate insecticide chlorpyrifos (CPF) is known to induce neurological effects, malformation and micronucleus formation, persistent developmental disorders, and maternal toxicity in rats and mice. The binding of chlorpyrifos with DNA to produce DNA adducts leads to an increasing social concern about the genotoxic risk of CPF in human, but CPF-induced cytotoxicity through DNA damage and cell apoptosis is not well understood. Here, we quantified the cytotoxicity and potential genotoxicity of CPF using the alkaline comet assay, γH2AX foci formation, and the DNA laddering assay in order to detect DNA damage and apoptosis in human HeLa and HEK293 cells in vitro. Drosophila S2 cells were used as a positive control. The alkaline comet assay showed that sublethal concentrations of CPF induced significant concentration-dependent increases in single-strand DNA breaks in the treated cells compared with the control. The percentage of γH2AX-positive HeLa cells revealed that CPF also causes DNA double-strand breaks in a time-dependent manner. Moreover, DNA fragmentation analysis demonstrated that exposure to CPF induced a significant concentration- and time-dependent increase in cell apoptosis. We conclude that CPF is a strongly genotoxic agent that induces DNA damage and cell apoptosis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Phosphorylation-Dependent Regulation of the DNA Damage Response of Adaptor Protein KIBRA in Cancer Cells.

PubMed

Mavuluri, Jayadev; Beesetti, Swarnalatha; Surabhi, Rohan; Kremerskothen, Joachim; Venkatraman, Ganesh; Rayala, Suresh K

2016-05-01

Multifunctional adaptor proteins encompassing various protein-protein interaction domains play a central role in the DNA damage response pathway. In this report, we show that KIBRA is a physiologically interacting reversible substrate of ataxia telangiectasia mutated (ATM) kinase. We identified the site of phosphorylation in KIBRA as threonine 1006, which is embedded within the serine/threonine (S/T) Q consensus motif, by site-directed mutagenesis, and we further confirmed the same with a phospho-(S/T) Q motif-specific antibody. Results from DNA repair functional assays such as the γ-H2AX assay, pulsed-field gel electrophoresis (PFGE), Comet assay, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and clonogenic cell survival assay using stable overexpression clones of wild-type (wt.) KIBRA and active (T1006E) and inactive (T1006A) KIBRA phosphorylation mutants showed that T1006 phosphorylation on KIBRA is essential for optimal DNA double-strand break repair in cancer cells. Further, results from stable retroviral short hairpin RNA-mediated knockdown (KD) clones of KIBRA and KIBRA knockout (KO) model cells generated by a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system showed that depleting KIBRA levels compromised the DNA repair functions in cancer cells upon inducing DNA damage. All these phenotypic events were reversed upon reconstitution of KIBRA into cells lacking KIBRA knock-in (KI) model cells. All these results point to the fact that phosphorylated KIBRA might be functioning as a scaffolding protein/adaptor protein facilitating the platform for further recruitment of other DNA damage response factors. In summary, these data demonstrate the imperative functional role of KIBRAper se(KIBRA phosphorylation at T1006 site as a molecular switch that regulates the DNA damage response, possibly via the nonhomologous end joining [NHEJ] pathway), suggesting that KIBRA could be a potential

Microcystin-LR induced DNA damage in human peripheral blood lymphocytes.

PubMed

Zegura, B; Gajski, G; Straser, A; Garaj-Vrhovac, V; Filipič, M

2011-12-24

Human exposure to microcystins, which are produced by freshwater cyanobacterial species, is of growing concern due to increasing appearance of cyanobacterial blooms as a consequence of global warming and increasing water eutrophication. Although microcystins are considered to be liver-specific, there is evidence that they may also affect other tissues. These substances have been shown to induce DNA damage in vitro and in vivo, but the mechanisms of their genotoxic activity remain unclear. In human peripheral blood lymphocytes (HPBLs) exposure to non-cytotoxic concentrations (0, 0.1, 1 and 10μg/ml) of microcystin-LR (MCLR) induced a dose- and time-dependent increase in DNA damage, as measured with the comet assay. Digestion of DNA from MCLR-treated HPBLs with purified formamidopyrimidine-DNA glycosylase (Fpg) displayed a greater number of DNA strand-breaks than non-digested DNA, confirming the evidence that MCLR induces oxidative DNA damage. With the cytokinesis-block micronucleus assay no statistically significant induction of micronuclei, nucleoplasmic bridges and nuclear buds was observed after a 24-h exposure to MCLR. At the molecular level, no changes in the expression of selected genes involved in the cellular response to DNA damage and oxidative stress were observed after a 4-h exposure to MCLR (1μg/ml). After 24h, DNA damage-responsive genes (p53, mdm2, gadd45a, cdkn1a), a gene involved in apoptosis (bax) and oxidative stress-responsive genes (cat, gpx1, sod1, gsr, gclc) were up-regulated. These results provide strong support that MCLR is an indirectly genotoxic agent, acting via induction of oxidative stress, and that lymphocytes are also the target of microcystin-induced toxicity. Copyright © 2011 Elsevier B.V. All rights reserved.

Sam68 Is Required for DNA Damage Responses via Regulating Poly(ADP-ribosyl)ation

PubMed Central

Hodgson, Andrea; Wier, Eric M.; Wen, Matthew G.; Kamenyeva, Olena; Xia, Xue; Koo, Lily Y.

2016-01-01

The rapid and robust synthesis of polymers of adenosine diphosphate (ADP)-ribose (PAR) chains, primarily catalyzed by poly(ADP-ribose) polymerase 1 (PARP1), is crucial for cellular responses to DNA damage. However, the precise mechanisms through which PARP1 is activated and PAR is robustly synthesized are not fully understood. Here, we identified Src-associated substrate during mitosis of 68 kDa (Sam68) as a novel signaling molecule in DNA damage responses (DDRs). In the absence of Sam68, DNA damage-triggered PAR production and PAR-dependent DNA repair signaling were dramatically diminished. With serial cellular and biochemical assays, we demonstrated that Sam68 is recruited to and significantly overlaps with PARP1 at DNA lesions and that the interaction between Sam68 and PARP1 is crucial for DNA damage-initiated and PARP1-conferred PAR production. Utilizing cell lines and knockout mice, we illustrated that Sam68-deleted cells and animals are hypersensitive to genotoxicity caused by DNA-damaging agents. Together, our findings suggest that Sam68 plays a crucial role in DDR via regulating DNA damage-initiated PAR production. PMID:27635653

Mus308 Processes Oxygen and Nitrogen Ethylation DNA Damage in Germ Cells of Drosophila

PubMed Central

Díaz-Valdés, Nancy; Comendador, Miguel A.; Sierra, L. María

2010-01-01

The D. melanogaster mus308 gene, highly conserved among higher eukaryotes, is implicated in the repair of cross-links and of O-ethylpyrimidine DNA damage, working in a DNA damage tolerance mechanism. However, despite its relevance, its possible role on the processing of different DNA ethylation damages is not clear. To obtain data on mutation frequency and on mutation spectra in mus308 deficient (mus308−) conditions, the ethylating agent diethyl sulfate (DES) was analysed in postmeiotic male germ cells. These data were compared with those corresponding to mus308 efficient conditions. Our results indicate that Mus308 is necessary for the processing of oxygen and N-ethylation damage, for the survival of fertilized eggs depending on the level of induced DNA damage, and for an influence of the DNA damage neighbouring sequence. These results support the role of mus308 in a tolerance mechanism linked to a translesion synthesis pathway and also to the alternative end-joinig system. PMID:20936147

Differences in DNA-damage in non-smoking men and women exposed to environmental tobacco smoke (ETS).

PubMed

Collier, Abby C; Dandge, Sachin D; Woodrow, James E; Pritsos, Chris A

2005-07-28

There is much data implicating environmental tobacco smoke (ETS) in the development and progression of disease, notably cancer, yet the mechanisms for this remain unclear. As ETS is both a pro-oxidant stressor and carcinogen, we investigated the relationship of ETS exposure to intracellular and serum levels of DNA-damage, both oxidative 8-hydroxy-2-deoxyguanosine (8OHdG) and general, in non-smokers from non-smoking households, occupationally exposed to ETS. General DNA-damage consisting of single and double strand breaks, alkali-labile sites and incomplete base-excision repair, increased significantly in a dose-dependent manner with ETS exposure in men (P=0.015, n=32, Pearson) but not women (P=0.736, n=17). Intracellular 8OHdG-DNA-damage and general DNA-damage were both greater in men than women (P=0.0005 and 0.016, respectively) but 8OHdG serum levels did not differ between the genders. Neither 8OHdG-DNA-damage nor serum levels correlated with increasing ETS exposure. This is the first study to demonstrate dose-dependent increases in DNA-damage from workplace ETS exposure. Perhaps most interesting was that despite equivalent ETS exposure, significantly greater DNA-damage occurred in men than women. These data may begin to provide a mechanistic rationale for the generally higher incidence of some diseases in males due to tobacco smoke and/or other genotoxic stressors.

Inhibition of the HDAC/Suv39/G9a pathway restores the expression of DNA damage-dependent major histocompatibility complex class I-related chain A and B in cancer cells.

PubMed

Nakajima, Nakako Izumi; Niimi, Atsuko; Isono, Mayu; Oike, Takahiro; Sato, Hiro; Nakano, Takashi; Shibata, Atsushi

2017-08-01

Immunotherapy is expected to be promising as a next generation cancer therapy. Immunoreceptors are often activated constitutively in cancer cells, however, such levels of ligand expression are not effectively recognized by the native immune system due to tumor microenvironmental adaptation. Studies have demonstrated that natural-killer group 2, member D (NKG2D), a major activating immunoreceptor, responds to DNA damage. The upregulation of major histocompatibility complex class I-related chain A and B (MICA/B) (members of NKG2D ligands) expression after DNA damage is associated with NK cell-mediated killing of cancer cells. However, the regulation of DNA damage-induced MICA/B expression has not been fully elucidated in the context of the types of cancer cell lines. In the present study, we found that MICA/B expression varied between cancer cell lines after DNA damage. Screening in terms of chromatin remodeling identified that inhibitors related to chromatin relaxation via post-translational modification on histone H3K9, i.e. HDAC, Suv39 or G9a inhibition, restored DNA damage-dependent MICA/B expression in insensitive cells. In addition, we revealed that the restored MICA/B expression was dependent on ATR as well as E2F1, a transcription factor. We further revealed that low‑dose treatment of an HDAC inhibitor was sufficient to restore MICA/B expression in insensitive cells. Finally, we demonstrated that HDAC inhibition restored DNA damage‑dependent cytotoxic NK activity against insensitive cells. Thus, the present study revealed that DNA damage‑dependent MICA/B expression in insensitive cancer cells can be restored by chromatin relaxation via the HDAC/Suv39/G9a pathway. Collectively, manipulation of chromatin status by therapeutic cancer drugs may potentiate the antitumor effect by enhancing immune activation following radiotherapy and DNA damage-associated chemotherapy.

UV Damage-Induced Phosphorylation of HBO1 Triggers CRL4DDB2-Mediated Degradation To Regulate Cell Proliferation

PubMed Central

Matsunuma, Ryoichi; Ohhata, Tatsuya; Kitagawa, Kyoko; Sakai, Satoshi; Uchida, Chiharu; Shiotani, Bunsyo; Matsumoto, Masaki; Nakayama, Keiichi I.; Ogura, Hiroyuki; Shiiya, Norihiko; Kitagawa, Masatoshi

2015-01-01

Histone acetyltransferase binding to ORC-1 (HBO1) is a critically important histone acetyltransferase for forming the prereplicative complex (pre-RC) at the replication origin. Pre-RC formation is completed by loading of the MCM2-7 heterohexameric complex, which functions as a helicase in DNA replication. HBO1 recruited to the replication origin by CDT1 acetylates histone H4 to relax the chromatin conformation and facilitates loading of the MCM complex onto replication origins. However, the acetylation status and mechanism of regulation of histone H3 at replication origins remain elusive. HBO1 positively regulates cell proliferation under normal cell growth conditions. Whether HBO1 regulates proliferation in response to DNA damage is poorly understood. In this study, we demonstrated that HBO1 was degraded after DNA damage to suppress cell proliferation. Ser50 and Ser53 of HBO1 were phosphorylated in an ATM/ATR DNA damage sensor-dependent manner after UV treatment. ATM/ATR-dependently phosphorylated HBO1 preferentially interacted with DDB2 and was ubiquitylated by CRL4DDB2. Replacement of endogenous HBO1 in Ser50/53Ala mutants maintained acetylation of histone H3K14 and impaired cell cycle regulation in response to UV irradiation. Our findings demonstrate that HBO1 is one of the targets in the DNA damage checkpoint. These results show that ubiquitin-dependent control of the HBO1 protein contributes to cell survival during UV irradiation. PMID:26572825

HIPK2 restricts SIRT1 activity upon severe DNA damage by a phosphorylation-controlled mechanism

PubMed Central

Conrad, E; Polonio-Vallon, T; Meister, M; Matt, S; Bitomsky, N; Herbel, C; Liebl, M; Greiner, V; Kriznik, B; Schumacher, S; Krieghoff-Henning, E; Hofmann, T G

2016-01-01

Upon severe DNA damage a cellular signalling network initiates a cell death response through activating tumour suppressor p53 in association with promyelocytic leukaemia (PML) nuclear bodies. The deacetylase Sirtuin 1 (SIRT1) suppresses cell death after DNA damage by antagonizing p53 acetylation. To facilitate efficient p53 acetylation, SIRT1 function needs to be restricted. How SIRT1 activity is regulated under these conditions remains largely unclear. Here we provide evidence that SIRT1 activity is limited upon severe DNA damage through phosphorylation by the DNA damage-responsive kinase HIPK2. We found that DNA damage provokes interaction of SIRT1 and HIPK2, which phosphorylates SIRT1 at Serine 682 upon lethal damage. Furthermore, upon DNA damage SIRT1 and HIPK2 colocalize at PML nuclear bodies, and PML depletion abrogates DNA damage-induced SIRT1 Ser682 phosphorylation. We show that Ser682 phosphorylation inhibits SIRT1 activity and impacts on p53 acetylation, apoptotic p53 target gene expression and cell death. Mechanistically, we found that DNA damage-induced SIRT1 Ser682 phosphorylation provokes disruption of the complex between SIRT1 and its activator AROS. Our findings indicate that phosphorylation-dependent restriction of SIRT1 activity by HIPK2 shapes the p53 response. PMID:26113041

DNA Damage Response, Redox Status and Hematopoiesis

PubMed Central

Weiss, Cary N.; Ito, Keisuke

2013-01-01

The ability of hematopoietic stem cells (HSCs) to self-renew and differentiate into progenitors is essential for homeostasis of the hematopoietic system. The longevity of HSCs makes them vulnerable to accumulating DNA damage, which may be leukemogenic or result in senescence and cell death. Additionally, the ability of HSCs to self-renew and differentiate allows DNA damage to spread throughout the hematologic system, leaving the organism vulnerable to disease. In this review we discuss cell fate decisions made in the face of DNA damage and other cellular stresses, and the role of reactive oxygen species in the long-term maintenance of HSCs and their DNA damage response. PMID:24041596

Effects of different levels of vitamin C on UV radiation-induced DNA damage

NASA Astrophysics Data System (ADS)

Zhou, Dianfeng; Heng, Hang; Ji, Kang; Ke, Weizhong

2005-05-01

The Raman spectra of DNA in different levels of vitamin C with 10- and 30-min ultraviolet (UV) radiations were reported. The intensity of UV radiation was 18.68 W/m2. The experimental results proved that vitamin C could alone prevent UV radiation from damaging DNA, but the effects depended on the concentration of vitamin C. When the concentration of vitamin C was about 0.08-0.4 mmol/L, vitamin C decreased UV radiation-induced DNA's damage. When the concentration of vitamin C exceeded 0.4 mmol/L, vitamin C accelerated DNA's damage instead. Maybe the reason is that when DNA in aqueous solution is radiated by UV, free radicals come into being, and vitamin C can scavenge free radicals, so vitamin C in lower concentration can protect DNA. The quantity of free radicals is finite, when vitamin C is superfluous, free radicals have been scavenged absolutely and vitamin C is residual. Vitamin C is a strong reductant. When the mixture of DNA and residual vitamin C is radiated by UV, vitamin C reacts with DNA. The more residual vitamin C and the longer time of UV radiation, the more DNA is damaged.

Close encounters for the first time: Helicase interactions with DNA damage.

PubMed

Khan, Irfan; Sommers, Joshua A; Brosh, Robert M

2015-09-01

DNA helicases are molecular motors that harness the energy of nucleoside triphosphate hydrolysis to unwinding structured DNA molecules that must be resolved during cellular replication, DNA repair, recombination, and transcription. In vivo, DNA helicases are expected to encounter a wide spectrum of covalent DNA modifications to the sugar phosphate backbone or the nitrogenous bases; these modifications can be induced by endogenous biochemical processes or exposure to environmental agents. The frequency of lesion abundance can vary depending on the lesion type. Certain adducts such as oxidative base modifications can be quite numerous, and their effects can be helix-distorting or subtle perturbations to DNA structure. Helicase encounters with specific DNA lesions and more novel forms of DNA damage will be discussed. We will also review the battery of assays that have been used to characterize helicase-catalyzed unwinding of damaged DNA substrates. Characterization of the effects of specific DNA adducts on unwinding by various DNA repair and replication helicases has proven to be insightful for understanding mechanistic and biological aspects of helicase function in cellular DNA metabolism. Published by Elsevier B.V.

The multifaceted influence of histone deacetylases on DNA damage signalling and DNA repair

PubMed Central

Roos, Wynand Paul; Krumm, Andrea

2016-01-01

Histone/protein deacetylases play multiple roles in regulating gene expression and protein activation and stability. Their deregulation during cancer initiation and progression cause resistance to therapy. Here, we review the role of histone deacetylases (HDACs) and the NAD+ dependent sirtuins (SIRTs) in the DNA damage response (DDR). These lysine deacetylases contribute to DNA repair by base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), non-homologous end joining (NHEJ), homologous recombination (HR) and interstrand crosslink (ICL) repair. Furthermore, we discuss possible mechanisms whereby these histone/protein deacetylases facilitate the switch between DNA double-strand break (DSB) repair pathways, how SIRTs play a central role in the crosstalk between DNA repair and cell death pathways due to their dependence on NAD+, and the influence of small molecule HDAC inhibitors (HDACi) on cancer cell resistance to genotoxin based therapies. Throughout the review, we endeavor to identify the specific HDAC targeted by HDACi leading to therapy sensitization. PMID:27738139

HSV-I and the cellular DNA damage response.

PubMed

Smith, Samantha; Weller, Sandra K

2015-04-01

Peter Wildy first observed genetic recombination between strains of HSV in 1955. At the time, knowledge of DNA repair mechanisms was limited, and it has only been in the last decade that particular DNA damage response (DDR) pathways have been examined in the context of viral infections. One of the first reports addressing the interaction between a cellular DDR protein and HSV-1 was the observation by Lees-Miller et al . that DNA-dependent protein kinase catalytic subunit levels were depleted in an ICP0-dependent manner during Herpes simplex virus 1 infection. Since then, there have been numerous reports describing the interactions between HSV infection and cellular DDR pathways. Due to space limitations, this review will focus predominantly on the most recent observations regarding how HSV navigates a potentially hostile environment to replicate its genome.

Mechanisms of DNA damage, repair and mutagenesis

PubMed Central

Chatterjee, Nimrat; Walker, Graham C.

2017-01-01

Living organisms are continuously exposed to a myriad of DNA damaging agents that can impact health and modulate disease-states. However, robust DNA repair and damage-bypass mechanisms faithfully protect the DNA by either removing or tolerating the damage to ensure an overall survival. Deviations in this fine-tuning are known to destabilize cellular metabolic homeostasis, as exemplified in diverse cancers where disruption or deregulation of DNA repair pathways results in genome instability. Because routinely used biological, physical and chemical agents impact human health, testing their genotoxicity and regulating their use have become important. In this introductory review, we will delineate mechanisms of DNA damage and the counteracting repair/tolerance pathways to provide insights into the molecular basis of genotoxicity in cells that lays the foundation for subsequent articles in this issue. PMID:28485537

Lyn tyrosine kinase promotes silencing of ATM-dependent checkpoint signaling during recovery from DNA double-strand breaks

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fukumoto, Yasunori, E-mail: fukumoto@faculty.chiba-u.jp; Kuki, Kazumasa; Morii, Mariko

2014-09-26

Highlights: • Inhibition of Src family kinases decreased γ-H2AX signal. • Inhibition of Src family increased ATM-dependent phosphorylation of Chk2 and Kap1. • shRNA-mediated knockdown of Lyn increased phosphorylation of Kap1 by ATM. • Ectopic expression of Src family kinase suppressed ATM-mediated Kap1 phosphorylation. • Src is involved in upstream signaling for inactivation of ATM signaling. - Abstract: DNA damage activates the DNA damage checkpoint and the DNA repair machinery. After initial activation of DNA damage responses, cells recover to their original states through completion of DNA repair and termination of checkpoint signaling. Currently, little is known about the processmore » by which cells recover from the DNA damage checkpoint, a process called checkpoint recovery. Here, we show that Src family kinases promote inactivation of ataxia telangiectasia mutated (ATM)-dependent checkpoint signaling during recovery from DNA double-strand breaks. Inhibition of Src activity increased ATM-dependent phosphorylation of Chk2 and Kap1. Src inhibition increased ATM signaling both in G2 phase and during asynchronous growth. shRNA knockdown of Lyn increased ATM signaling. Src-dependent nuclear tyrosine phosphorylation suppressed ATM-mediated Kap1 phosphorylation. These results suggest that Src family kinases are involved in upstream signaling that leads to inactivation of the ATM-dependent DNA damage checkpoint.« less

Role of Growth Arrest and DNA Damage–inducible α in Akt Phosphorylation and Ubiquitination after Mechanical Stress-induced Vascular Injury

PubMed Central

Mitra, Sumegha; Sammani, Saad; Wang, Ting; Boone, David L.; Meyer, Nuala J.; Dudek, Steven M.; Moreno-Vinasco, Liliana; Garcia, Joe G. N.

2011-01-01

Rationale: The stress-induced growth arrest and DNA damage–inducible α (GADD45a) gene is up-regulated by mechanical stress with GADD45a knockout (GADD45a−/−) mice demonstrating both increased susceptibility to ventilator-induced lung injury (VILI) and reduced levels of the cell survival and vascular permeability signaling effector (Akt). However, the functional role of GADD45a in the pathogenesis of VILI is unknown. Objectives: We sought to define the role of GADD45a in the regulation of Akt activation induced by mechanical stress. Methods: VILI-challenged GADD45a−/− mice were administered a constitutively active Akt1 vector and injury was assessed by bronchoalveolar lavage cell counts and protein levels. Human pulmonary artery endothelial cells (EC) were exposed to 18% cyclic stretch (CS) under conditions of GADD45a silencing and used for immunoprecipitation, Western blotting or immunofluoresence. EC were also transfected with mutant ubiquitin vectors to characterize site-specific Akt ubiquitination. DNA methylation was measured using methyl-specific polymerase chain reaction assay. Measurements and Main Results: Studies exploring the linkage of GADD45a with mechanical stress and Akt regulation revealed VILI-challenged GADD45a−/− mice to have significantly reduced lung injury on overexpression of Akt1 transgene. Increased mechanical stress with 18% CS in EC induced Akt phosphorylation via E3 ligase tumor necrosis factor receptor–associated factor 6 (TRAF6)–mediated Akt K63 ubiquitination resulting in Akt trafficking and activation at the membrane. GADD45a is essential to this process because GADD45a-silenced endothelial cells and GADD45a−/− mice exhibited increased Akt K48 ubiquitination leading to proteasomal degradation. These events involve loss of ubiquitin carboxyl terminal hydrolase 1 (UCHL1), a deubiquitinating enzyme that normally removes K48 polyubiquitin chains bound to Akt thus promoting Akt K63 ubiquitination. Loss of GADD45a

Ubiquitin-dependent and independent roles of SUMO in proteostasis.

PubMed

Liebelt, Frauke; Vertegaal, Alfred C O

2016-08-01

Cellular proteomes are continuously undergoing alterations as a result of new production of proteins, protein folding, and degradation of proteins. The proper equilibrium of these processes is known as proteostasis, implying that proteomes are in homeostasis. Stress conditions can affect proteostasis due to the accumulation of misfolded proteins as a result of overloading the degradation machinery. Proteostasis is affected in neurodegenerative diseases like Alzheimer's disease, Parkinson's disease, and multiple polyglutamine disorders including Huntington's disease. Owing to a lack of proteostasis, neuronal cells build up toxic protein aggregates in these diseases. Here, we review the role of the ubiquitin-like posttranslational modification SUMO in proteostasis. SUMO alone contributes to protein homeostasis by influencing protein signaling or solubility. However, the main contribution of SUMO to proteostasis is the ability to cooperate with, complement, and balance the ubiquitin-proteasome system at multiple levels. We discuss the identification of enzymes involved in the interplay between SUMO and ubiquitin, exploring the complexity of this crosstalk which regulates proteostasis. These enzymes include SUMO-targeted ubiquitin ligases and ubiquitin proteases counteracting these ligases. Additionally, we review the role of SUMO in brain-related diseases, where SUMO is primarily investigated because of its role during formation of aggregates, either independently or in cooperation with ubiquitin. Detailed understanding of the role of SUMO in these diseases could lead to novel treatment options. Copyright © 2016 the American Physiological Society.

Noise Induced DNA Damage Within the Auditory Nerve.

PubMed

Guthrie, O'neil W

2017-03-01

An understanding of the molecular pathology that underlies noise induced neurotoxicity is a prerequisite to the design of targeted therapies. The objective of the current experiment was to determine whether or not DNA damage is part of the pathophysiologic sequela of noise induced neurotoxicity. The experiment consisted of 41 hooded Long-Evans rats (2 month old males) that were randomized into control and noise exposed groups. Both the control and the noise group followed the same time schedule and therefore started and ended the experiment together. The noise dose consisted of a 6000 Hz noise band at 105 dB SPL. Temporal bones from both groups were harvested, and immunohistochemistry was used to identify neurons with DNA damage. Quantitative morphometric analyses was then employed to determine the level of DNA damage. Neural action potentials were recorded to assess the functional impact of noise induced DNA damage. Immunohistochemical reactions revealed that the noise exposure precipitated DNA damage within the nucleus of auditory neurons. Quantitative morphometry confirmed the noise induced increase in DNA damage levels and the precipitation of DNA damage was associated with a significant loss of nerve sensitivity. Therefore, DNA damage is part of the molecular pathology that drives noise induced neurotoxicity. Anat Rec, 300:520-526, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

A CAF-1–PCNA-Mediated Chromatin Assembly Pathway Triggered by Sensing DNA Damage

PubMed Central

Moggs, Jonathan G.; Grandi, Paola; Quivy, Jean-Pierre; Jónsson, Zophonías O.; Hübscher, Ulrich; Becker, Peter B.; Almouzni, Geneviève

2000-01-01

Sensing DNA damage is crucial for the maintenance of genomic integrity and cell cycle progression. The participation of chromatin in these events is becoming of increasing interest. We show that the presence of single-strand breaks and gaps, formed either directly or during DNA damage processing, can trigger the propagation of nucleosomal arrays. This nucleosome assembly pathway involves the histone chaperone chromatin assembly factor 1 (CAF-1). The largest subunit (p150) of this factor interacts directly with proliferating cell nuclear antigen (PCNA), and critical regions for this interaction on both proteins have been mapped. To isolate proteins specifically recruited during DNA repair, damaged DNA linked to magnetic beads was used. The binding of both PCNA and CAF-1 to this damaged DNA was dependent on the number of DNA lesions and required ATP. Chromatin assembly linked to the repair of single-strand breaks was disrupted by depletion of PCNA from a cell-free system. This defect was rescued by complementation with recombinant PCNA, arguing for role of PCNA in mediating chromatin assembly linked to DNA repair. We discuss the importance of the PCNA–CAF-1 interaction in the context of DNA damage processing and checkpoint control. PMID:10648606

Transcription and DNA Damage: Holding Hands or Crossing Swords?

PubMed

D'Alessandro, Giuseppina; d'Adda di Fagagna, Fabrizio

2017-10-27

Transcription has classically been considered a potential threat to genome integrity. Collision between transcription and DNA replication machinery, and retention of DNA:RNA hybrids, may result in genome instability. On the other hand, it has been proposed that active genes repair faster and preferentially via homologous recombination. Moreover, while canonical transcription is inhibited in the proximity of DNA double-strand breaks, a growing body of evidence supports active non-canonical transcription at DNA damage sites. Small non-coding RNAs accumulate at DNA double-strand break sites in mammals and other organisms, and are involved in DNA damage signaling and repair. Furthermore, RNA binding proteins are recruited to DNA damage sites and participate in the DNA damage response. Here, we discuss the impact of transcription on genome stability, the role of RNA binding proteins at DNA damage sites, and the function of small non-coding RNAs generated upon damage in the signaling and repair of DNA lesions. Copyright © 2016 Elsevier Ltd. All rights reserved.

A Perturbed Ubiquitin Landscape Distinguishes Between Ubiquitin in Trafficking and in Proteolysis*

PubMed Central

Ziv, Inbal; Matiuhin, Yulia; Kirkpatrick, Donald S.; Erpapazoglou, Zoi; Leon, Sebastien; Pantazopoulou, Marina; Kim, Woong; Gygi, Steven P.; Haguenauer-Tsapis, Rosine; Reis, Noa; Glickman, Michael H.; Kleifeld, Oded

2011-01-01

Any of seven lysine residues on ubiquitin can serve as the base for chain-extension, resulting in a sizeable spectrum of ubiquitin modifications differing in chain length or linkage type. By optimizing a procedure for rapid lysis, we charted the profile of conjugated cellular ubiquitin directly from whole cell extract. Roughly half of conjugated ubiquitin (even at high molecular weights) was nonextended, consisting of monoubiquitin modifications and chain terminators (endcaps). Of extended ubiquitin, the primary linkages were via Lys48 and Lys63. All other linkages were detected, contributing a relatively small portion that increased at lower molecular weights. In vivo expression of lysineless ubiquitin (K0 Ub) perturbed the ubiquitin landscape leading to elevated levels of conjugated ubiquitin, with a higher mono-to-poly ratio. Affinity purification of these trapped conjugates identified a comprehensive list of close to 900 proteins including novel targets. Many of the proteins enriched by K0 ubiquitination were membrane-associated, or involved in cellular trafficking. Prime among them are components of the ESCRT machinery and adaptors of the Rsp5 E3 ubiquitin ligase. Ubiquitin chains associated with these substrates were enriched for Lys63 linkages over Lys48, indicating that K0 Ub is unevenly distributed throughout the ubiquitinome. Biological assays validated the interference of K0 Ub with protein trafficking and MVB sorting, minimally affecting Lys48-dependent turnover of proteasome substrates. We conclude that despite the shared use of the ubiquitin molecule, the two branches of the ubiquitin machinery—the ubiquitin-proteasome system and the ubiquitin trafficking system—were unevenly perturbed by expression of K0 ubiquitin. PMID:21427232

DNA Damage Signals and Space Radiation Risk

NASA Technical Reports Server (NTRS)

Cucinotta, Francis A.

2011-01-01

Space radiation is comprised of high-energy and charge (HZE) nuclei and protons. The initial DNA damage from HZE nuclei is qualitatively different from X-rays or gamma rays due to the clustering of damage sites which increases their complexity. Clustering of DNA damage occurs on several scales. First there is clustering of single strand breaks (SSB), double strand breaks (DSB), and base damage within a few to several hundred base pairs (bp). A second form of damage clustering occurs on the scale of a few kbp where several DSB?s may be induced by single HZE nuclei. These forms of damage clusters do not occur at low to moderate doses of X-rays or gamma rays thus presenting new challenges to DNA repair systems. We review current knowledge of differences that occur in DNA repair pathways for different types of radiation and possible relationships to mutations, chromosomal aberrations and cancer risks.

Inactivating UBE2M impacts the DNA damage response and genome integrity involving multiple cullin ligases.

PubMed

Cukras, Scott; Morffy, Nicholas; Ohn, Takbum; Kee, Younghoon

2014-01-01

Protein neddylation is involved in a wide variety of cellular processes. Here we show that the DNA damage response is perturbed in cells inactivated with an E2 Nedd8 conjugating enzyme UBE2M, measured by RAD51 foci formation kinetics and cell based DNA repair assays. UBE2M knockdown increases DNA breakages and cellular sensitivity to DNA damaging agents, further suggesting heightened genomic instability and defective DNA repair activity. Investigating the downstream Cullin targets of UBE2M revealed that silencing of Cullin 1, 2, and 4 ligases incurred significant DNA damage. In particular, UBE2M knockdown, or defective neddylation of Cullin 2, leads to a blockade in the G1 to S progression and is associated with delayed S-phase dependent DNA damage response. Cullin 4 inactivation leads to an aberrantly high DNA damage response that is associated with increased DNA breakages and sensitivity of cells to DNA damaging agents, suggesting a DNA repair defect is associated. siRNA interrogation of key Cullin substrates show that CDT1, p21, and Claspin are involved in elevated DNA damage in the UBE2M knockdown cells. Therefore, UBE2M is required to maintain genome integrity by activating multiple Cullin ligases throughout the cell cycle.

Direct Detection and Sequencing of Damaged DNA Bases

PubMed Central

2011-01-01

Products of various forms of DNA damage have been implicated in a variety of important biological processes, such as aging, neurodegenerative diseases, and cancer. Therefore, there exists great interest to develop methods for interrogating damaged DNA in the context of sequencing. Here, we demonstrate that single-molecule, real-time (SMRT®) DNA sequencing can directly detect damaged DNA bases in the DNA template - as a by-product of the sequencing method - through an analysis of the DNA polymerase kinetics that are altered by the presence of a modified base. We demonstrate the sequencing of several DNA templates containing products of DNA damage, including 8-oxoguanine, 8-oxoadenine, O6-methylguanine, 1-methyladenine, O4-methylthymine, 5-hydroxycytosine, 5-hydroxyuracil, 5-hydroxymethyluracil, or thymine dimers, and show that these base modifications can be readily detected with single-modification resolution and DNA strand specificity. We characterize the distinct kinetic signatures generated by these DNA base modifications. PMID:22185597

Direct detection and sequencing of damaged DNA bases.

PubMed

Clark, Tyson A; Spittle, Kristi E; Turner, Stephen W; Korlach, Jonas

2011-12-20

Products of various forms of DNA damage have been implicated in a variety of important biological processes, such as aging, neurodegenerative diseases, and cancer. Therefore, there exists great interest to develop methods for interrogating damaged DNA in the context of sequencing. Here, we demonstrate that single-molecule, real-time (SMRT®) DNA sequencing can directly detect damaged DNA bases in the DNA template - as a by-product of the sequencing method - through an analysis of the DNA polymerase kinetics that are altered by the presence of a modified base. We demonstrate the sequencing of several DNA templates containing products of DNA damage, including 8-oxoguanine, 8-oxoadenine, O6-methylguanine, 1-methyladenine, O4-methylthymine, 5-hydroxycytosine, 5-hydroxyuracil, 5-hydroxymethyluracil, or thymine dimers, and show that these base modifications can be readily detected with single-modification resolution and DNA strand specificity. We characterize the distinct kinetic signatures generated by these DNA base modifications.

Repair of Clustered Damage and DNA Polymerase Iota.

PubMed

Belousova, E A; Lavrik, O I

2015-08-01

Multiple DNA lesions occurring within one or two turns of the DNA helix known as clustered damage are a source of double-stranded DNA breaks, which represent a serious threat to the cells. Repair of clustered lesions is accomplished in several steps. If a clustered lesion contains oxidized bases, an individual DNA lesion is repaired by the base excision repair (BER) mechanism involving a specialized DNA polymerase after excising DNA damage. Here, we investigated DNA synthesis catalyzed by DNA polymerase iota using damaged DNA templates. Two types of DNA substrates were used as model DNAs: partial DNA duplexes containing breaks of different length, and DNA duplexes containing 5-formyluracil (5-foU) and uracil as a precursor of apurinic/apyrimidinic sites (AP) in opposite DNA strands. For the first time, we showed that DNA polymerase iota is able to catalyze DNA synthesis using partial DNA duplexes having breaks of different length as substrates. In addition, we found that DNA polymerase iota could catalyze DNA synthesis during repair of clustered damage via the BER system by using both undamaged and 5-foU-containing templates. We found that hPCNA (human proliferating cell nuclear antigen) increased efficacy of DNA synthesis catalyzed by DNA polymerase iota.

SOX9 is targeted for proteasomal degradation by the E3 ligase FBW7 in response to DNA damage

PubMed Central

Hong, Xuehui; Liu, Wenyu; Song, Ruipeng; Shah, Jamie J.; Feng, Xing; Tsang, Chi Kwan; Morgan, Katherine M.; Bunting, Samuel F.; Inuzuka, Hiroyuki; Zheng, X. F. Steven; Shen, Zhiyuan; Sabaawy, Hatem E.; Liu, LianXin; Pine, Sharon R.

2016-01-01

SOX9 encodes a transcription factor that governs cell fate specification throughout development and tissue homeostasis. Elevated SOX9 is implicated in the genesis and progression of human tumors by increasing cell proliferation and epithelial-mesenchymal transition. We found that in response to UV irradiation or genotoxic chemotherapeutics, SOX9 is actively degraded in various cancer types and in normal epithelial cells, through a pathway independent of p53, ATM, ATR and DNA-PK. SOX9 is phosphorylated by GSK3β, facilitating the binding of SOX9 to the F-box protein FBW7α, an E3 ligase that functions in the DNA damage response pathway. The binding of FBW7α to the SOX9 K2 domain at T236-T240 targets SOX9 for subsequent ubiquitination and proteasomal destruction. Exogenous overexpression of SOX9 after genotoxic stress increases cell survival. Our findings reveal a novel regulatory mechanism for SOX9 stability and uncover a unique function of SOX9 in the cellular response to DNA damage. This new mechanism underlying a FBW7-SOX9 axis in cancer could have implications in therapy resistance. PMID:27566146

DNA Damage and Repair: Relevance to Mechanisms of Neurodegeneration

PubMed Central

Martin, Lee J.

2008-01-01

DNA damage is a form of cell stress and injury that has been implicated in the pathogenesis of many neurologic disorders, including amyotrophic lateral sclerosis, Alzheimer disease, Down syndrome, Parkinson disease, cerebral ischemia, and head trauma. However, most data reveal only associations, and the role for DNA damage in direct mechanisms of neurodegeneration is vague with respect to being a definitive upstream cause of neuron cell death, rather than a consequence of the degeneration. Although neurons seem inclined to develop DNA damage during oxidative stress, most of the existing work on DNA damage and repair mechanisms has been done in the context of cancer biology using cycling non-neuronal cells but not nondividing (i.e. postmitotic) neurons. Nevertheless, the identification of mutations in genes that encode proteins that function in DNA repair and DNA damage response in human hereditary DNA repair deficiency syndromes and ataxic disorders is establishing a mechanistic precedent that clearly links DNA damage and DNA repair abnormalities with progressive neurodegeneration. This review summarizes DNA damage and repair mechanisms and their potential relevance to the evolution of degeneration in postmitotic neurons. PMID:18431258

Catch the live show: Visualizing damaged DNA in vivo.

PubMed

Oshidari, Roxanne; Mekhail, Karim

2018-06-01

The health of an organism is intimately linked to its ability to repair damaged DNA. Importantly, DNA repair processes are highly dynamic. This highlights the necessity of characterizing DNA repair in live cells. Advanced genome editing and imaging approaches allow us to visualize damaged DNA and its associated factors in real time. Here, we summarize both established and recent methods that are used to induce DNA damage and visualize damaged DNA and its repair in live cells. Copyright © 2018 Elsevier Inc. All rights reserved.

USP48 restrains resection by site-specific cleavage of the BRCA1 ubiquitin mark from H2A.

PubMed

Uckelmann, Michael; Densham, Ruth M; Baas, Roy; Winterwerp, Herrie H K; Fish, Alexander; Sixma, Titia K; Morris, Joanna R

2018-01-15

BRCA1-BARD1-catalyzed ubiquitination of histone H2A is an important regulator of the DNA damage response, priming chromatin for repair by homologous recombination. However, no specific deubiquitinating enzymes (DUBs) are known to antagonize this function. Here we identify ubiquitin specific protease-48 (USP48) as a H2A DUB, specific for the C-terminal BRCA1 ubiquitination site. Detailed biochemical analysis shows that an auxiliary ubiquitin, an additional ubiquitin that itself does not get cleaved, modulates USP48 activity, which has possible implications for its regulation in vivo. In cells we reveal that USP48 antagonizes BRCA1 E3 ligase function and in BRCA1-proficient cells loss of USP48 results in positioning 53BP1 further from the break site and in extended resection lengths. USP48 repression confers a survival benefit to cells treated with camptothecin and its activity acts to restrain gene conversion and mutagenic single-strand annealing. We propose that USP48 promotes genome stability by antagonizing BRCA1 E3 ligase function.

Ubiquitin fusion expression and tissue-dependent targeting of hG-CSF in transgenic tobacco

PubMed Central

2011-01-01

Background Human granulocyte colony-stimulating factor (hG-CSF) is an important human cytokine which has been widely used in oncology and infection protection. To satisfy clinical needs, expression of recombinant hG-CSF has been studied in several organisms, including rice cell suspension culture and transient expression in tobacco leaves, but there was no published report on its expression in stably transformed plants which can serve as a more economical expression platform with potential industrial application. Results In this study, hG-CSF expression was investigated in transgenic tobacco leaves and seeds in which the accumulation of hG-CSF could be enhanced through fusion with ubiquitin by up to 7 fold in leaves and 2 fold in seeds, leading to an accumulation level of 2.5 mg/g total soluble protein (TSP) in leaves and 1.3 mg/g TSP in seeds, relative to hG-CSF expressed without a fusion partner. Immunoblot analysis showed that ubiquitin was processed from the final protein product, and ubiquitination was up-regulated in all transgenic plants analyzed. Driven by CaMV 35S promoter and phaseolin signal peptide, hG-CSF was observed to be secreted into apoplast in leaves but deposited in protein storage vacuole (PSV) in seeds, indicating that targeting of the hG-CSF was tissue-dependent in transgenic tobacco. Bioactivity assay showed that hG-CSF expressed in both seeds and leaves was bioactive to support the proliferation of NFS-60 cells. Conclusions In this study, the expression of bioactive hG-CSF in transgenic plants was improved through ubiquitin fusion strategy, demonstrating that protein expression can be enhanced in both plant leaves and seeds through fusion with ubiquitin and providing a typical case of tissue-dependent expression of recombinant protein in transgenic plants. PMID:21985646

The Molecular Origin of the MMR-dependent Apoptosis Pathway from Dynamics Analysis of MutSα-DNA Complexes

PubMed Central

Negureanu, Lacramioara; Salsbury, Freddie R.

2012-01-01

The cellular response to DNA damage signaling by MMR proteins is incompletely understood. It is generally accepted that MMR-dependent apoptosis pathway in response to DNA damage detection is independent of MMR's DNA repair function. In this study we investigate correlated motions in response to the binding of mismatched and PCL DNA fragments by MutSα, as derived from 50 ns molecular dynamics simulations. The protein dynamics in response to the mismatched and damaged DNA recognition suggests that MutSα signals their recognition through independent pathways providing evidence for the molecular origin of the MMR-dependent apoptosis. MSH2 subunit is indicated to play a key role in signaling both mismatched and damaged DNA recognition; localized and collective motions within the protein allow identifying sites on the MSH2 surface possible involved in recruiting proteins responsible for downstream events. Unlike in the mismatch complex, predicted key communication sites specific for the damage recognition are on the list of known cancer causing mutations or deletions. This confirms MSH2's role in signaling DNA-damage induced apoptosis and suggests that defects in MMR alone is sufficient to trigger tumorigenesis, supporting the experimental evidence that MMR-damage response function could protect from the early occurrence of tumors. Identifying these particular communication sites may have implications for the treatment of cancers that are not defective for MMR, but are unable to function optimally for MMR-dependent responses following DNA damage such as the case of resistance to cisplatin. PMID:22712459

BAG3 Expression in Glioblastoma Cells Promotes Accumulation of Ubiquitinated Clients in an Hsp70-dependent Manner*

PubMed Central

Gentilella, Antonio; Khalili, Kamel

2011-01-01

Disposal of damaged proteins and protein aggregates is a prerequisite for the maintenance of cellular homeostasis and impairment of this disposal can lead to a broad range of pathological conditions, most notably in brain-associated disorders including Parkinson and Alzheimer diseases, and cancer. In this respect, the Protein Quality Control (PQC) pathway plays a central role in the clearance of damaged proteins. The Hsc/Hsp70-co-chaperone BAG3 has been described as a new and critical component of the PQC in several cellular contexts. For example, the expression of BAG3 in the rodent brain correlates with the engagement of protein degradation machineries in response to proteotoxic stress. Nevertheless, little is known about the molecular events assisted by BAG3. Here we show that ectopic expression of BAG3 in glioblastoma cells leads to the activation of an HSF1-driven stress response, as attested by transcriptional activation of BAG3 and Hsp70. BAG3 overexpression determines an accumulation of ubiquitinated proteins and this event requires the N-terminal region, WW domain of BAG3 and the association of BAG3 with Hsp70. The ubiquitination mainly occurs on BAG3-client proteins and the inhibition of proteasomal activity results in a further accumulation of ubiquitinated clients. At the cellular level, overexpression of BAG3 in glioblastoma cell lines, but not in non-glial cells, results in a remarkable decrease in colony formation capacity and this effect is reverted when the binding of BAG3 to Hsp70 is impaired. These observations provide the first evidence for an involvement of BAG3 in the ubiquitination and turnover of its partners. PMID:21233200

Electronic cigarette aerosols suppress cellular antioxidant defenses and induce significant oxidative DNA damage

PubMed Central

Ganapathy, Vengatesh; Manyanga, Jimmy; Brame, Lacy; McGuire, Dehra; Sadhasivam, Balaji; Floyd, Evan; Rubenstein, David A.; Ramachandran, Ilangovan; Wagener, Theodore

2017-01-01

Background Electronic cigarette (EC) aerosols contain unique compounds in addition to toxicants and carcinogens traditionally found in tobacco smoke. Studies are warranted to understand the public health risks of ECs. Objective The aim of this study was to determine the genotoxicity and the mechanisms induced by EC aerosol extracts on human oral and lung epithelial cells. Methods Cells were exposed to EC aerosol or mainstream smoke extracts and DNA damage was measured using the primer anchored DNA damage detection assay (q-PADDA) and 8-oxo-dG ELISA assay. Cell viability, reactive oxygen species (ROS) and total antioxidant capacity (TAC) were measured using standard methods. mRNA and protein expression were evaluated by RT-PCR and western blot, respectively. Results EC aerosol extracts induced DNA damage in a dose-dependent manner, but independently of nicotine concentration. Overall, EC aerosol extracts induced significantly less DNA damage than mainstream smoke extracts, as measured by q-PADDA. However, the levels of oxidative DNA damage, as indicated by the presence of 8-oxo-dG, a highly mutagenic DNA lesion, were similar or slightly higher after exposure to EC aerosol compared to mainstream smoke extracts. Mechanistically, while exposure to EC extracts significantly increased ROS, it decreased TAC as well as the expression of 8-oxoguanine DNA glycosylase (OGG1), an enzyme essential for the removal of oxidative DNA damage. Conclusions Exposure to EC aerosol extracts suppressed the cellular antioxidant defenses and led to significant DNA damage. These findings emphasize the urgent need to investigate the potential long-term cancer risk of exposure to EC aerosol for vapers and the general public. PMID:28542301

CDK1 promotes nascent DNA synthesis and induces resistance of cancer cells to DNA-damaging therapeutic agents

PubMed Central

Liao, Hongwei; Ji, Fang; Geng, Xinwei; Xing, Meichun; Li, Wen; Chen, Zhihua; Shen, Huahao; Ying, Songmin

2017-01-01

Cyclin dependent kinase 1 (CDK1) is essential for cell viability and plays a vital role in many biological events including cell cycle control, DNA damage repair, and checkpoint activation. Here, we identify an unanticipated role for CDK1 in promoting nascent DNA synthesis during S-phase. We report that a short duration of CDK1 inhibition, which does not perturb cell cycle progression, triggers a replication-associated DNA damage response (DDR). This DDR is associated with a disruption of replication fork progression and leads to genome instability. Moreover, we show that compromised CDK1 activity dramatically increases the efficacy of chemotherapeutic agents that kill cancer cells through perturbing DNA replication, including Olaparib, an FDA approved PARP inhibitor. Our study has revealed an important role for CDK1 in the DNA replication program, and suggests that the therapeutic targeting CDK1 may be a novel approach for combination chemotherapy. PMID:29207595

DNA Damage and Pulmonary Hypertension

PubMed Central

Ranchoux, Benoît; Meloche, Jolyane; Paulin, Roxane; Boucherat, Olivier; Provencher, Steeve; Bonnet, Sébastien

2016-01-01

Pulmonary hypertension (PH) is defined by a mean pulmonary arterial pressure over 25 mmHg at rest and is diagnosed by right heart catheterization. Among the different groups of PH, pulmonary arterial hypertension (PAH) is characterized by a progressive obstruction of distal pulmonary arteries, related to endothelial cell dysfunction and vascular cell proliferation, which leads to an increased pulmonary vascular resistance, right ventricular hypertrophy, and right heart failure. Although the primary trigger of PAH remains unknown, oxidative stress and inflammation have been shown to play a key role in the development and progression of vascular remodeling. These factors are known to increase DNA damage that might favor the emergence of the proliferative and apoptosis-resistant phenotype observed in PAH vascular cells. High levels of DNA damage were reported to occur in PAH lungs and remodeled arteries as well as in animal models of PH. Moreover, recent studies have demonstrated that impaired DNA-response mechanisms may lead to an increased mutagen sensitivity in PAH patients. Finally, PAH was linked with decreased breast cancer 1 protein (BRCA1) and DNA topoisomerase 2-binding protein 1 (TopBP1) expression, both involved in maintaining genome integrity. This review aims to provide an overview of recent evidence of DNA damage and DNA repair deficiency and their implication in PAH pathogenesis. PMID:27338373

Inhibition of Protein Ubiquitination by Paraquat and 1-Methyl-4-Phenylpyridinium Impairs Ubiquitin-Dependent Protein Degradation Pathways.

PubMed

Navarro-Yepes, Juliana; Anandhan, Annadurai; Bradley, Erin; Bohovych, Iryna; Yarabe, Bo; de Jong, Annemieke; Ovaa, Huib; Zhou, You; Khalimonchuk, Oleh; Quintanilla-Vega, Betzabet; Franco, Rodrigo

2016-10-01

Intracytoplasmic inclusions of protein aggregates in dopaminergic cells (Lewy bodies) are the pathological hallmark of Parkinson's disease (PD). Ubiquitin (Ub), alpha (α)-synuclein, p62/sequestosome 1, and oxidized proteins are the major components of Lewy bodies. However, the mechanisms involved in the impairment of misfolded/oxidized protein degradation pathways in PD are still unclear. PD is linked to mitochondrial dysfunction and environmental pesticide exposure. In this work, we evaluated the effects of the pesticide paraquat (PQ) and the mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP(+)) on Ub-dependent protein degradation pathways. No increase in the accumulation of Ub-bound proteins or aggregates was observed in dopaminergic cells (SK-N-SH) treated with PQ or MPP(+), or in mice chronically exposed to PQ. PQ decreased Ub protein content, but not its mRNA transcription. Protein synthesis inhibition with cycloheximide depleted Ub levels and potentiated PQ-induced cell death. The inhibition of proteasomal activity by PQ was found to be a late event in cell death progression and had neither effect on the toxicity of either MPP(+) or PQ, nor on the accumulation of oxidized sulfenylated, sulfonylated (DJ-1/PARK7 and peroxiredoxins), and carbonylated proteins induced by PQ. PQ- and MPP(+)-induced Ub protein depletion prompted the dimerization/inactivation of the Ub-binding protein p62 that regulates the clearance of ubiquitinated proteins by autophagy. We confirmed that PQ and MPP(+) impaired autophagy flux and that the blockage of autophagy by the overexpression of a dominant-negative form of the autophagy protein 5 (dnAtg5) stimulated their toxicity, but there was no additional effect upon inhibition of the proteasome. PQ induced an increase in the accumulation of α-synuclein in dopaminergic cells and membrane-associated foci in yeast cells. Our results demonstrate that the inhibition of protein ubiquitination by PQ and MPP(+) is involved in the

Inhibition of protein ubiquitination by paraquat and 1-methyl-4-phenylpyridinium impairs ubiquitin-dependent protein degradation pathways

PubMed Central

Navarro-Yepes, Juliana; Anandhan, Annadurai; Bradley, Erin; Bohovych, Iryna; Yarabe, Bo; de Jong, Annemieke; Ovaa, Huib; Zhou, You; Khalimonchuk, Oleh; Quintanilla-Vega, Betzabet; Franco, Rodrigo

2016-01-01

Intracytoplasmic inclusions of protein aggregates in dopaminergic cells (Lewy bodies) are the pathological hallmark of Parkinson’s disease (PD). Ubiquitin (Ub), alpha [α]-synuclein, p62/sequestosome 1 and oxidized proteins are major components of Lewy bodies. However, the mechanisms involved in the impairment of misfolded/oxidized protein degradation pathways in PD are still unclear. PD is linked to mitochondrial dysfunction and environmental pesticide exposure. In this work, we evaluated the effect of the pesticide paraquat (PQ) and the mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP+) on Ub-dependent protein degradation pathways. No increase in the accumulation of Ub-bound proteins or aggregates was observed in dopaminergic cells (SK-N-SH) treated with PQ or MPP+, or in mice chronically exposed to PQ. PQ decreased Ub protein content, but not its mRNA transcription. Protein synthesis inhibition with cycloheximide depleted Ub levels and potentiated PQ–induced cell death. Inhibition of proteasomal activity by PQ was found to be a late event in cell death progression, and had no effect on either the toxicity of MPP+ or PQ, or the accumulation of oxidized sulfenylated, sulfonylated (DJ-1/PARK7 and peroxiredoxins) and carbonylated proteins induced by PQ. PQ- and MPP+-induced Ub protein depletion prompted the dimerization/inactivation of the Ub-binding protein p62 that regulates the clearance of ubiquitinated proteins by autophagic. We confirmed that PQ and MPP+ impaired autophagy flux, and that the blockage of autophagy by the overexpression of a dominant-negative form of the autophagy protein 5 (dnAtg5) stimulated their toxicity, but there was no additional effect upon inhibition of the proteasome. PQ induced an increase in the accumulation of α-synuclein in dopaminergic cells and membrane associated foci in yeast cells. Our results demonstrate that inhibition of protein ubiquitination by PQ and MPP+ is involved in the dysfunction of Ub-dependent protein

DNA Replication Arrest and DNA Damage Responses Induced by Alkylating Minor Groove Binders

DTIC Science & Technology

2001-05-01

We are interested in the molecular mechanisms involved in DNA replication arrest by the S phase DNA damage checkpoints. Using in vitro simian virus...40 DNA replication assays, we have found three factors that directly contribute to DNA damage-induced DNA replication arrest: Replication Protein A...trans-acting inhibitors. RPA is the major eukaryotic single-stranded DNA binding protein required for DNA replication , repair and recombination. Upon DNA

The Linear ubiquitin chain assembly complex acts as a liver tumor suppressor and inhibits hepatocyte apoptosis and hepatitis.

PubMed

Shimizu, Yutaka; Peltzer, Nieves; Sevko, Alexandra; Lafont, Elodie; Sarr, Aida; Draberova, Helena; Walczak, Henning

2017-06-01

Linear ubiquitination is a key posttranslational modification that regulates immune signaling and cell death pathways, notably tumor necrosis factor receptor 1 (TNFR1) signaling. The only known enzyme complex capable of forming linear ubiquitin chains under native conditions to date is the linear ubiquitin chain assembly complex, of which the catalytic core component is heme-oxidized iron regulatory protein 2 ubiquitin ligase-1-interacting protein (HOIP). To understand the underlying mechanisms of maintenance of liver homeostasis and the role of linear ubiquitination specifically in liver parenchymal cells, we investigated the physiological role of HOIP in the liver parenchyma. To do so, we created mice harboring liver parenchymal cell-specific deletion of HOIP (Hoip Δhep mice) by crossing Hoip-floxed mice with albumin-Cre mice. HOIP deficiency in liver parenchymal cells triggered tumorigenesis at 18 months of age preceded by spontaneous hepatocyte apoptosis and liver inflammation within the first month of life. In line with the emergence of inflammation, Hoip Δhep mice displayed enhanced liver regeneration and DNA damage. In addition, consistent with increased apoptosis, HOIP-deficient hepatocytes showed enhanced caspase activation and endogenous formation of a death-inducing signaling complex which activated caspase-8. Unexpectedly, exacerbated caspase activation and apoptosis were not dependent on TNFR1, whereas ensuing liver inflammation and tumorigenesis were promoted by TNFR1 signaling. The linear ubiquitin chain assembly complex serves as a previously undescribed tumor suppressor in the liver, restraining TNFR1-independent apoptosis in hepatocytes which, in its absence, is causative of TNFR1-mediated inflammation, resulting in hepatocarcinogenesis. (Hepatology 2017;65:1963-1978). © 2017 The Authors. Hepatology published by Wiley Periodicals, Inc., on behalf of the American Association for the Study of Liver Diseases.

E2 Ubiquitin-conjugating Enzyme, UBE2C Gene, Is Reciprocally Regulated by Wild-type and Gain-of-Function Mutant p53.

PubMed

Bajaj, Swati; Alam, Sk Kayum; Roy, Kumar Singha; Datta, Arindam; Nath, Somsubhra; Roychoudhury, Susanta

2016-07-01

Spindle assembly checkpoint governs proper chromosomal segregation during mitosis to ensure genomic stability. At the cellular level, this event is tightly regulated by UBE2C, an E2 ubiquitin-conjugating enzyme that donates ubiquitin to the anaphase-promoting complex/cyclosome. This, in turn, facilitates anaphase-onset by ubiquitin-mediated degradation of mitotic substrates. UBE2C is an important marker of chromosomal instability and has been associated with malignant growth. However, the mechanism of its regulation is largely unexplored. In this study, we report that UBE2C is transcriptionally activated by the gain-of-function (GOF) mutant p53, although it is transcriptionally repressed by wild-type p53. We showed that wild-type p53-mediated inhibition of UBE2C is p21-E2F4-dependent and GOF mutant p53-mediated transactivation of UBE2C is NF-Y-dependent. We further explored that DNA damage-induced wild-type p53 leads to spindle assembly checkpoint arrest by repressing UBE2C, whereas mutant p53 causes premature anaphase exit by increasing UBE2C expression in the presence of 5-fluorouracil. Identification of UBE2C as a target of wild-type and GOF mutant p53 further highlights the contribution of p53 in regulation of spindle assembly checkpoint. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

DNA damage under simulated extraterrestrial conditions in bacteriophage T7

NASA Astrophysics Data System (ADS)

Fekete, A.; Módos, K.; Hegedüs, M.; Kovács, G.; Rontó, Gy.; Péter, Á.; Lammer, H.; Panitz, C.

The experiment "Phage and Uracil response" will be accommodated in the EXPOSE facility of the International Space Station. Its objective is to examine and quantify the effect of specific space conditions on nucleic acid models, especially on bacteriophage T7 and isolated T7 DNA thin films. In order to define the environmental and technical requirements of the EXPOSE, the samples were subjected to the experiment verification test (EVT). During EVT, the samples were exposed to vacuum (10 -4-10 -6 Pa) and polychromatic UV-radiation (200-400 nm) in air, in inert atmosphere, as well as in simulated space vacuum. The effect of extreme temperature in vacuum and the influence of temperature fluctuations around 0 °C were also studied. The total intraphage/isolated DNA damage was determined by quantitative PCR using 555 and 3826 bp fragments of T7 DNA. The type of the damage was resolved using a combination of enzymatic probes and neutral and alkaline agarose gel electrophoresis; the structural/chemical effects were analyzed by spectroscopic and microscopical methods. We obtained substantial evidence that DNA lesions accumulate throughout exposure, but the amount of damage depends on the thickness of the layers. According to our preliminary results, the damages by exposure to conditions of dehydration and UV-irradiation are larger than the sum of vacuum alone, or radiation alone case, suggesting a synergistic action of space vacuum and UV radiation with DNA being the critical target.

Preterm newborns show slower repair of oxidative damage and paternal smoking associated DNA damage.

PubMed

Vande Loock, Kim; Ciardelli, Roberta; Decordier, Ilse; Plas, Gina; Haumont, Dominique; Kirsch-Volders, Micheline

2012-09-01

Newborns have to cope with hypoxia during delivery and a sudden increase in oxygen at birth. Oxygen will partly be released as reactive oxygen species having the potential to cause damage to DNA and proteins. In utero, increase of most (non)-enzymatic antioxidants occurs during last weeks of gestation, making preterm neonates probably more sensitive to oxidative stress. Moreover, it has been hypothesized that oxidative stress might be the common etiological factor for certain neonatal diseases in preterm infants. The aim of this study was to assess background DNA damage; in vitro H(2)O(2) induced oxidative DNA damage and repair capacity (residual DNA damage) in peripheral blood mononucleated cells from 25 preterm newborns and their mothers. In addition, demographic data were taken into account and repair capacity of preterm was compared with full-term newborns. Multivariate linear regression analysis revealed that preterm infants from smoking fathers have higher background DNA damage levels than those from non-smoking fathers, emphasizing the risk of paternal smoking behaviour for the progeny. Significantly higher residual DNA damage found after 15-min repair in preterm children compared to their mothers and higher residual DNA damage after 2 h compared to full-term newborns suggest a slower DNA repair capacity in preterm children. In comparison with preterm infants born by caesarean delivery, preterm infants born by vaginal delivery do repair more slowly the in vitro induced oxidative DNA damage. Final impact of passive smoking and of the slower DNA repair activity of preterm infants need to be confirmed in a larger study population combining transgenerational genetic and/or epigenetic effects, antioxidant levels, genotypes, repair enzyme efficiency/levels and infant morbidity.

DNA DAMAGE QUANTITATION BY ALKALINE GEL ELECTROPHORESIS.

DOE Office of Scientific and Technical Information (OSTI.GOV)

SUTHERLAND,B.M.; BENNETT,P.V.; SUTHERLAND, J.C.

2004-03-24

Physical and chemical agents in the environment, those used in clinical applications, or encountered during recreational exposures to sunlight, induce damages in DNA. Understanding the biological impact of these agents requires quantitation of the levels of such damages in laboratory test systems as well as in field or clinical samples. Alkaline gel electrophoresis provides a sensitive (down to {approx} a few lesions/5Mb), rapid method of direct quantitation of a wide variety of DNA damages in nanogram quantities of non-radioactive DNAs from laboratory, field, or clinical specimens, including higher plants and animals. This method stems from velocity sedimentation studies of DNAmore » populations, and from the simple methods of agarose gel electrophoresis. Our laboratories have developed quantitative agarose gel methods, analytical descriptions of DNA migration during electrophoresis on agarose gels (1-6), and electronic imaging for accurate determinations of DNA mass (7-9). Although all these components improve sensitivity and throughput of large numbers of samples (7,8,10), a simple version using only standard molecular biology equipment allows routine analysis of DNA damages at moderate frequencies. We present here a description of the methods, as well as a brief description of the underlying principles, required for a simplified approach to quantitation of DNA damages by alkaline gel electrophoresis.« less

Increased mitochondrial DNA deletions and copy number in transfusion-dependent thalassemia

PubMed Central

Calloway, Cassandra

2016-01-01

BACKGROUND. Iron overload is the primary cause of morbidity in transfusion-dependent thalassemia. Increase in iron causes mitochondrial dysfunction under experimental conditions, but the occurrence and significance of mitochondrial damage is not understood in patients with thalassemia. METHODS. Mitochondrial DNA (mtDNA) to nuclear DNA copy number (Mt/N) and frequency of the common 4977-bp mitochondrial deletion (ΔmtDNA4977) were quantified using a quantitative PCR assay on whole blood samples from 38 subjects with thalassemia who were receiving regular transfusions. RESULTS. Compared with healthy controls, Mt/N and ΔmtDNA4977 frequency were elevated in thalassemia (P = 0.038 and P < 0.001, respectively). ΔmtDNA4977 was increased in the presence of either liver iron concentration > 15 mg/g dry-weight or splenectomy, with the highest levels observed in subjects who had both risk factors (P = 0.003). Myocardial iron (MRI T2* < 20 ms) was present in 0%, 22%, and 46% of subjects with ΔmtDNA4977 frequency < 20, 20–40, and > 40/1 × 107 mtDNA, respectively (P = 0.025). Subjects with Mt/N values below the group median had significantly lower Matsuda insulin sensitivity index (5.76 ± 0.53) compared with the high Mt/N group (9.11 ± 0.95, P = 0.008). CONCLUSION. Individuals with transfusion-dependent thalassemia demonstrate age-related increase in mtDNA damage in leukocytes. These changes are markedly amplified by splenectomy and are associated with extrahepatic iron deposition. Elevated mtDNA damage in blood cells may predict the risk of iron-associated organ damage in thalassemia. FUNDING. This project was supported by Children’s Hospital & Research Center Oakland Institutional Research Award and by the National Center for Advancing Translational Sciences, NIH, through UCSF-CTSI grant UL1 TR000004. PMID:27583305

Mitochondrial DNA repair and damage tolerance.

PubMed

Stein, Alexis; Sia, Elaine A

2017-01-01

The accurate maintenance of mitochondrial DNA (mtDNA) is required in order for eukaryotic cells to assemble a functional electron transport chain. This independently-maintained genome relies on nuclear-encoded proteins that are imported into the mitochondria to carry out replication and repair processes. Decades of research has made clear that mitochondria employ robust and varied mtDNA repair and damage tolerance mechanisms in order to ensure the proper maintenance of the mitochondrial genome. This review focuses on our current understanding of mtDNA repair and damage tolerance pathways including base excision repair, mismatch repair, homologous recombination, non-homologous end joining, translesion synthesis and mtDNA degradation in both yeast and mammalian systems.

Ubiquitin-like protein UBL5 promotes the functional integrity of the Fanconi anemia pathway.

PubMed

Oka, Yasuyoshi; Bekker-Jensen, Simon; Mailand, Niels

2015-05-12

Ubiquitin and ubiquitin-like proteins (UBLs) function in a wide array of cellular processes. UBL5 is an atypical UBL that does not form covalent conjugates with cellular proteins and which has a known role in modulating pre-mRNA splicing. Here, we report an unexpected involvement of human UBL5 in promoting the function of the Fanconi anemia (FA) pathway for repair of DNA interstrand crosslinks (ICLs), mediated by a specific interaction with the central FA pathway component FANCI. UBL5-deficient cells display spliceosome-independent reduction of FANCI protein stability, defective FANCI function in response to DNA damage and hypersensitivity to ICLs. By mapping the sequence determinants underlying UBL5-FANCI binding, we generated separation-of-function mutants to demonstrate that key aspects of FA pathway function, including FANCI-FANCD2 heterodimerization, FANCD2 and FANCI monoubiquitylation and maintenance of chromosome stability after ICLs, are compromised when the UBL5-FANCI interaction is selectively inhibited by mutations in either protein. Together, our findings establish UBL5 as a factor that promotes the functionality of the FA DNA repair pathway. © 2015 The Authors.

The ubiquitin-proteasome system is required for African swine fever replication.

PubMed

Barrado-Gil, Lucía; Galindo, Inmaculada; Martínez-Alonso, Diego; Viedma, Sergio; Alonso, Covadonga

2017-01-01

Several viruses manipulate the ubiquitin-proteasome system (UPS) to initiate a productive infection. Determined viral proteins are able to change the host's ubiquitin machinery and some viruses even encode their own ubiquitinating or deubiquitinating enzymes. African swine fever virus (ASFV) encodes a gene homologous to the E2 ubiquitin conjugating (UBC) enzyme. The viral ubiquitin-conjugating enzyme (UBCv1) is expressed throughout ASFV infection and accumulates at late times post infection. UBCv is also present in the viral particle suggesting that the ubiquitin-proteasome pathway could play an important role at early ASFV infection. We determined that inhibition of the final stage of the ubiquitin-proteasome pathway blocked a post-internalization step in ASFV replication in Vero cells. Under proteasome inhibition, ASF viral genome replication, late gene expression and viral production were severely reduced. Also, ASFV enhanced proteasome activity at late times and the accumulation of polyubiquitinated proteins surrounding viral factories. Core-associated and/or viral proteins involved in DNA replication may be targets for the ubiquitin-proteasome pathway that could possibly assist virus uncoating at final core breakdown and viral DNA release. At later steps, polyubiquitinated proteins at viral factories could exert regulatory roles in cell signaling.

Ubiquitin recognition by FAAP20 expands the complex interface beyond the canonical UBZ domain

PubMed Central

Wojtaszek, Jessica L.; Wang, Su; Kim, Hyungjin; Wu, Qinglin; D'Andrea, Alan D.; Zhou, Pei

2014-01-01

FAAP20 is an integral component of the Fanconi anemia core complex that mediates the repair of DNA interstrand crosslinks. The ubiquitin-binding capacity of the FAAP20 UBZ is required for recruitment of the Fanconi anemia complex to interstrand DNA crosslink sites and for interaction with the translesion synthesis machinery. Although the UBZ–ubiquitin interaction is thought to be exclusively encapsulated within the ββα module of UBZ, we show that the FAAP20–ubiquitin interaction extends beyond such a canonical zinc-finger motif. Instead, ubiquitin binding by FAAP20 is accompanied by transforming a disordered tail C-terminal to the UBZ of FAAP20 into a rigid, extended β-loop that latches onto the complex interface of the FAAP20 UBZ and ubiquitin, with the invariant C-terminal tryptophan emanating toward I44Ub for enhanced binding specificity and affinity. Substitution of the C-terminal tryptophan with alanine in FAAP20 not only abolishes FAAP20–ubiquitin binding in vitro, but also causes profound cellular hypersensitivity to DNA interstrand crosslink lesions in vivo, highlighting the indispensable role of the C-terminal tail of FAAP20, beyond the compact zinc finger module, toward ubiquitin recognition and Fanconi anemia complex-mediated DNA interstrand crosslink repair. PMID:25414354

Stress-induced DNA Damage biomarkers: Applications and limitations

NASA Astrophysics Data System (ADS)

Nikitaki, Zacharenia; Hellweg, Christine; Georgakilas, Alexandros; Ravanat, Jean-Luc

2015-06-01

A variety of environmental stresses like chemicals, UV and ionizing radiation and organism’s endogenous processes like replication stress and metabolism can lead to the generation of reactive oxygen and nitrogen species (ROS/RNS) that can attack cellular vital components like DNA, proteins and lipid membranes. Among them, much attention has been focused on DNA since DNA damages play a role in several biological disorders and aging processes. Thus, DNA damage can be used as a biomarker in a reliable and accurate way to quantify for example radiation exposure and can indicate its possible long term effects and cancer risk. Based on the type of DNA lesions detected one can hypothesize on the most probable mechanisms involved in the formation of these lesions for example in the case of UV and ionizing radiation (e.g. X- or α-, γ-rays, energetic ions, neutrons). In this review we describe the most accepted chemical pathways for DNA damage induction and the different types of DNA lesions, i.e. single, complex DNA lesions etc. that can be used as biomarkers. We critically compare DNA damage detection methods and their limitations. In addition to such DNA damage products, we suggest possible gene inductions that can be used to characterize responses to different types of stresses i.e. radiation, oxidative and replication stress, based on bioinformatic approaches and stringent meta-analysis of literature data.

Mechanisms of free radical-induced damage to DNA.

PubMed

Dizdaroglu, Miral; Jaruga, Pawel

2012-04-01

Endogenous and exogenous sources cause free radical-induced DNA damage in living organisms by a variety of mechanisms. The highly reactive hydroxyl radical reacts with the heterocyclic DNA bases and the sugar moiety near or at diffusion-controlled rates. Hydrated electron and H atom also add to the heterocyclic bases. These reactions lead to adduct radicals, further reactions of which yield numerous products. These include DNA base and sugar products, single- and double-strand breaks, 8,5'-cyclopurine-2'-deoxynucleosides, tandem lesions, clustered sites and DNA-protein cross-links. Reaction conditions and the presence or absence of oxygen profoundly affect the types and yields of the products. There is mounting evidence for an important role of free radical-induced DNA damage in the etiology of numerous diseases including cancer. Further understanding of mechanisms of free radical-induced DNA damage, and cellular repair and biological consequences of DNA damage products will be of outmost importance for disease prevention and treatment.

Activation of DNA damage repair pathways by murine polyomavirus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heiser, Katie; Nicholas, Catherine; Garcea, Robert

Nuclear replication of DNA viruses activates DNA damage repair (DDR) pathways, which are thought to detect and inhibit viral replication. However, many DNA viruses also depend on these pathways in order to optimally replicate their genomes. We investigated the relationship between murine polyomavirus (MuPyV) and components of DDR signaling pathways including CHK1, CHK2, H2AX, ATR, and DNAPK. We found that recruitment and retention of DDR proteins at viral replication centers was independent of H2AX, as well as the viral small and middle T-antigens. Additionally, infectious virus production required ATR kinase activity, but was independent of CHK1, CHK2, or DNAPK signaling.more » ATR inhibition did not reduce the total amount of viral DNA accumulated, but affected the amount of virus produced, indicating a defect in virus assembly. These results suggest that MuPyV may utilize a subset of DDR proteins or non-canonical DDR signaling pathways in order to efficiently replicate and assemble. -- Highlights: •Murine polyomavirus activates and recruits DNA damage repair (DDR) proteins to replication centers. •Large T-antigen mediates recruitment of DDR proteins to viral replication centers. •Inhibition or knockout of CHK1, CHK2, DNA-PK or H2AX do not affect viral titers. •Inhibition of ATR activity reduces viral titers, but not viral DNA accumulation.« less

The tomato DWD motif-containing protein DDI1 interacts with the CUL4–DDB1-based ubiquitin ligase and plays a pivotal role in abiotic stress responses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miao, Min; School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009; Department of Plant, Soil and Entomological Sciences, University of Idaho, Moscow, ID 83844-2339

2014-08-08

Highlights: • We identify DDI1 as a DAMAGED DNA BINDING PROTEIN1 (DDB1)-interacting protein. • DDI1 interacts with the CUL4–DDB1-based ubiquitin ligase in the nucleus. • DDI1 plays a positive role in regulating abiotic stress response in tomato. - Abstract: CULLIN4(CUL4)–DAMAGED DNA BINDING PROTEIN1 (DDB1)-based ubiquitin ligase plays significant roles in multiple physiological processes via ubiquitination-mediated degradation of relevant target proteins. The DDB1–CUL4-associated factor (DCAF) acts as substrate receptor in the CUL4–DDB1 ubiquitin ligase complex and determines substrate specificity. In this study, we identified a tomato (Solanum lycopersicum) DDB1-interacting (DDI1) protein as a DCAF protein involved in response to abiotic stresses,more » including UV radiation, high salinity and osmotic stress. Co-immunoprecipitation and bimolecular fluorescence complementation assay indicated that DDI1 associates with CUL4–DDB1 in the nucleus. Quantitative RT-PCR analysis indicated the DDI1 gene is induced by salt, mannitol and UV-C treatment. Moreover, transgenic tomato plants with overexpression or knockdown of the DDI1 gene exhibited enhanced or attenuated tolerance to salt/mannitol/UV-C, respectively. Thus, our data suggest that DDI1 functions as a substrate receptor of the CUL4–DDB1 ubiquitin ligase, positively regulating abiotic stress response in tomato.« less

Serine is the major residue for ADP-ribosylation upon DNA damage

PubMed Central

Dauben, Helen

2018-01-01

Poly(ADP-ribose) polymerases (PARPs) are a family of enzymes that synthesise ADP-ribosylation (ADPr), a reversible modification of proteins that regulates many different cellular processes. Several mammalian PARPs are known to regulate the DNA damage response, but it is not clear which amino acids in proteins are the primary ADPr targets. Previously, we reported that ARH3 reverses the newly discovered type of ADPr (ADPr on serine residues; Ser-ADPr) and developed tools to analyse this modification (Fontana et al., 2017). Here, we show that Ser-ADPr represents the major fraction of ADPr synthesised after DNA damage in mammalian cells and that globally Ser-ADPr is dependent on HPF1, PARP1 and ARH3. In the absence of HPF1, glutamate/aspartate becomes the main target residues for ADPr. Furthermore, we describe a method for site-specific validation of serine ADP-ribosylated substrates in cells. Our study establishes serine as the primary form of ADPr in DNA damage signalling. PMID:29480802

Mitochondrial DNA damage is associated with damage accrual and disease duration in patients with Systemic Lupus Erythematosus

PubMed Central

López-López, Linnette; Nieves-Plaza, Mariely; Castro, María del R.; Font, Yvonne M.; Torres-Ramos, Carlos; Vilá, Luis M.; Ayala-Peña, Sylvette

2014-01-01

Objective To determine the extent of mitochondrial DNA (mtDNA) damage in systemic lupus erythematosus (SLE) patients compared to healthy subjects and to determine the factors associated with mtDNA damage among SLE patients. Methods A cross-sectional study was performed in 86 SLE patients (per American College of Rheumatology classification criteria) and 86 healthy individuals matched for age and gender. Peripheral blood mononuclear cells (PBMCs) were collected from subjects to assess the relative amounts of mtDNA damage. Quantitative polymerase chain reaction assay was used to measure the frequency of mtDNA lesions and mtDNA abundance. Socioeconomic-demographic features, clinical manifestations, pharmacologic treatment, disease activity, and damage accrual were determined. Statistical analyses were performed using t test, pairwise correlation, and Pearson’s chi-square test (or Fisher’s exact test) as appropriate. Results Among SLE patients, 93.0% were women. The mean (SD) age was 38.0 (10.4) years and the mean (SD) disease duration was 8.7 (7.5) years. SLE patients exhibited increased levels of mtDNA damage as shown by higher levels of mtDNA lesions and decreased mtDNA abundance as compared to healthy individuals. There was a negative correlation between disease damage and mtDNA abundance and a positive correlation between mtDNA lesions and disease duration. No association was found between disease activity and mtDNA damage. Conclusion PBMCs from SLE patients exhibited more mtDNA damage compared to healthy subjects. Higher levels of mtDNA damage were observed among SLE patients with major organ involvement and damage accrual. These results suggest that mtDNA damage have a potential role in the pathogenesis of SLE. PMID:24899636

Posttranslational Modification of HOIP Blocks Toll-Like Receptor 4-Mediated Linear-Ubiquitin-Chain Formation

PubMed Central

Bowman, James; Rodgers, Mary A.; Shi, Mude; Amatya, Rina; Hostager, Bruce; Iwai, Kazuhiro; Gao, Shou-Jiang

2015-01-01

ABSTRACT Linear ubiquitination is an atypical posttranslational modification catalyzed by the linear-ubiquitin-chain assembly complex (LUBAC), containing HOIP, HOIL-1L, and Sharpin. LUBAC facilitates NF-κB activation and inflammation upon receptor stimulation by ligating linear ubiquitin chains to critical signaling molecules. Indeed, linear-ubiquitination-dependent signaling is essential to prevent pyogenic bacterial infections that can lead to death. While linear ubiquitination is essential for intracellular receptor signaling upon microbial infection, this response must be measured and stopped to avoid tissue damage and autoimmunity. While LUBAC is activated upon bacterial stimulation, the mechanisms regulating LUBAC activity in response to bacterial stimuli have remained elusive. We demonstrate that LUBAC activity itself is downregulated through ubiquitination, specifically, ubiquitination of the catalytic subunit HOIP at the carboxyl-terminal lysine 1056. Ubiquitination of Lys1056 dynamically altered HOIP conformation, resulting in the suppression of its catalytic activity. Consequently, HOIP Lys1056-to-Arg mutation led not only to persistent LUBAC activity but also to prolonged NF-κB activation induced by bacterial lipopolysaccharide-mediated Toll-like receptor 4 (TLR4) stimulation, whereas it showed no effect on NF-κB activation induced by CD40 stimulation. This study describes a novel posttranslational regulation of LUBAC-mediated linear ubiquitination that is critical for specifically directing TLR4-mediated NF-κB activation. PMID:26578682

SPOC1 modulates DNA repair by regulating key determinants of chromatin compaction and DNA damage response

PubMed Central

Mund, Andreas; Schubert, Tobias; Staege, Hannah; Kinkley, Sarah; Reumann, Kerstin; Kriegs, Malte; Fritsch, Lauriane; Battisti, Valentine; Ait-Si-Ali, Slimane; Hoffbeck, Anne-Sophie; Soutoglou, Evi; Will, Hans

2012-01-01

Survival time-associated plant homeodomain (PHD) finger protein in Ovarian Cancer 1 (SPOC1, also known as PHF13) is known to modulate chromatin structure and is essential for testicular stem-cell differentiation. Here we show that SPOC1 is recruited to DNA double-strand breaks (DSBs) in an ATM-dependent manner. Moreover, SPOC1 localizes at endogenous repair foci, including OPT domains and accumulates at large DSB repair foci characteristic for delayed repair at heterochromatic sites. SPOC1 depletion enhances the kinetics of ionizing radiation-induced foci (IRIF) formation after γ-irradiation (γ-IR), non-homologous end-joining (NHEJ) repair activity, and cellular radioresistance, but impairs homologous recombination (HR) repair. Conversely, SPOC1 overexpression delays IRIF formation and γH2AX expansion, reduces NHEJ repair activity and enhances cellular radiosensitivity. SPOC1 mediates dose-dependent changes in chromatin association of DNA compaction factors KAP-1, HP1-α and H3K9 methyltransferases (KMT) GLP, G9A and SETDB1. In addition, SPOC1 interacts with KAP-1 and H3K9 KMTs, inhibits KAP-1 phosphorylation and enhances H3K9 trimethylation. These findings provide the first evidence for a function of SPOC1 in DNA damage response (DDR) and repair. SPOC1 acts as a modulator of repair kinetics and choice of pathways. This involves its dose-dependent effects on DNA damage sensors, repair mediators and key regulators of chromatin structure. PMID:23034801

Triplex technology in studies of DNA damage, DNA repair, and mutagenesis.

PubMed

Mukherjee, Anirban; Vasquez, Karen M

2011-08-01

Triplex-forming oligonucleotides (TFOs) can bind to the major groove of homopurine-homopyrimidine stretches of double-stranded DNA in a sequence-specific manner through Hoogsteen hydrogen bonding to form DNA triplexes. TFOs by themselves or conjugated to reactive molecules can be used to direct sequence-specific DNA damage, which in turn results in the induction of several DNA metabolic activities. Triplex technology is highly utilized as a tool to study gene regulation, molecular mechanisms of DNA repair, recombination, and mutagenesis. In addition, TFO targeting of specific genes has been exploited in the development of therapeutic strategies to modulate DNA structure and function. In this review, we discuss advances made in studies of DNA damage, DNA repair, recombination, and mutagenesis by using triplex technology to target specific DNA sequences. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Intestinal tuft cells regulate the ATM mediated DNA Damage response via Dclk1 dependent mechanism for crypt restitution following radiation injury.

PubMed

Chandrakesan, Parthasarathy; May, Randal; Weygant, Nathaniel; Qu, Dongfeng; Berry, William L; Sureban, Sripathi M; Ali, Naushad; Rao, Chinthalapally; Huycke, Mark; Bronze, Michael S; Houchen, Courtney W

2016-11-23

Crypt epithelial survival and regeneration after injury require highly coordinated complex interplay between resident stem cells and diverse cell types. The function of Dclk1 expressing tuft cells regulating intestinal epithelial DNA damage response for cell survival/self-renewal after radiation-induced injury is unclear. Intestinal epithelial cells (IECs) were isolated and purified and utilized for experimental analysis. We found that small intestinal crypts of Villin Cre ;Dclk1 f/f mice were hypoplastic and more apoptotic 24 h post-total body irradiation, a time when stem cell survival is p53-independent. Injury-induced ATM mediated DNA damage response, pro-survival genes, stem cell markers, and self-renewal ability for survival and restitution were reduced in the isolated intestinal epithelial cells. An even greater reduction in these signaling pathways was observed 3.5 days post-TBI, when peak crypt regeneration occurs. We found that interaction with Dclk1 is critical for ATM and COX2 activation in response to injury. We determined that Dclk1 expressing tuft cells regulate the whole intestinal epithelial cells following injury through paracrine mechanism. These findings suggest that intestinal tuft cells play an important role in regulating the ATM mediated DNA damage response, for epithelial cell survival/self-renewal via a Dclk1 dependent mechanism, and these processes are indispensable for restitution and function after severe radiation-induced injury.

Requirement of the Saccharomyces cerevisiae APN1 Gene for the Repair of Mitochondrial DNA Alkylation Damage

PubMed Central

Acevedo-Torres, Karina; Fonseca-Williams, Sharon; Ayala-Torres, Sylvette; Torres-Ramos, Carlos A.

2010-01-01

The Saccharomyces cerevisiae APN1 gene that participates in base excision repair has been localized both in the nucleus and the mitochondria. APN1 deficient cells (apn1Δ) show increased mutation frequencies in mitochondrial DNA (mtDNA) suggesting that APN1 is also important for mtDNA stability. To understand APN1-dependent mtDNA repair processes we studied the formation and repair of mtDNA lesions in cells exposed to methyl methanesulfonate (MMS). We show that MMS induces mtDNA damage in a dose-dependent fashion and that deletion of the APN1 gene enhances the susceptibility of mtDNA to MMS. Repair kinetic experiments demonstrate that in wild-type cells (WT) it takes 4 hr to repair the damage induced by 0.1% MMS, whereas in the apn1Δ strain there is a lag in mtDNA repair that results in significant differences in the repair capacity between the two yeast strains. Analysis of lesions in nuclear DNA (nDNA) after treatment with 0.1% MMS shows a significant difference in the amount of nDNA lesions between WT and apn1Δ cells. Interestingly, comparisons between nDNA and mtDNA damage show that nDNA is more sensitive to the effects of MMS treatment. However, both strains are able to repair the nDNA lesions, contrary to mtDNA repair, which is compromised in the apn1Δ mutant strain. Therefore, although nDNA is more sensitive than mtDNA to the effects of MMS, deletion of APN1 has a stronger phenotype in mtDNA repair than in nDNA. These results highlight the prominent role of APN1 in the repair of environmentally induced mtDNA damage. PMID:19197988

DNA damage-induced nuclear translocation of Apaf-1 is mediated by nucleoporin Nup107

PubMed Central

Jagot-Lacoussiere, Léonard; Faye, Audrey; Bruzzoni-Giovanelli, Heriberto; Villoutreix, Bruno O; Rain, Jean-Christophe; Poyet, Jean-Luc

2015-01-01

Beside its central role in the mitochondria-dependent cell death pathway, the apoptotic protease activating factor 1 (Apaf-1) is involved in the DNA damage response through cell-cycle arrest induced by genotoxic stress. This non-apoptotic function requires a nuclear translocation of Apaf-1 during the G1-to-S transition. However, the mechanisms that trigger the nuclear accumulation of Apaf-1 upon DNA damage remain to be investigated. Here we show that the main 4 isoforms of Apaf-1 can undergo nuclear translocation and restore Apaf-1 deficient MEFs cell cycle arrest in the S phase following genotoxic stress through activation of Chk-1. Interestingly, DNA damage-dependent nuclear accumulation of Apaf-1 occurs independently of p53 and the retinoblastoma (pRb) pathway. We demonstrated that Apaf-1 associates with the nucleoporin Nup107 and this association is necessary for Apaf-1 nuclear import. The CED-4 domain of Apaf-1 directly binds to the central domain of Nup107 in an ATR-regulated, phosphorylation-dependent manner. Interestingly, expression of the Apaf-1-interacting domain of Nup107 interfered with Apaf-1 nuclear translocation upon genotoxic stress, resulting in a marked reduction of Chk-1 activation and cell cycle arrest. Thus, our results confirm the crucial role of Apaf-1 nuclear relocalization in mediating cell-cycle arrest induced by genotoxic stress and implicate Nup107 as a critical regulator of the DNA damage-induced intra-S phase checkpoint response. PMID:25695197

DNA Damage Dependence on the Subcellular Distribution of Low-Energy Beta Emitters

NASA Astrophysics Data System (ADS)

Cutaia, Claudia; Alloni, Daniele; Mariotti, Luca; Friedland, Werner; Ottolenghi, Andrea

One of the main issues of low-energy internal emitters is related to the short ranges of beta particles, compared to the dimensions of the biological targets (e.g. the cell nucleus). Also depending on the chemical form, the radionuclide may be more concentrated in the cytoplasm of the target cell (in our calculations a human fibroblast in interphase) and consequently the conventional dosimetry may overestimate the dose to the nucleus; whereas if the radionuclide is more concentrated in the nuclei of the cells there is a risk of underestimating the nucleus dose. The computer code PARTRAC was modified to calculate the energy depositions in the nucleus and the DNA damage for different relative concentrations of the radionuclide in the nucleus and in the cytoplasm. The nuclides considered in the simulations were Tritium (the electrons emitted due to the β - decay have an average energy of 5.7 keV, corresponding to an average range of 0.42 µm) and Nickel-63 (the electrons emitted have an average energy of 17 keV corresponding to an average range of 5 µm). In the case of Tritium, the dose in the nucleus due the tracks generated outside this region is 15% of the average dose in the cell, whereas in the case of Nickel-63 the dose in the nucleus resulted to be 64% of the average dose in the cell. The distributions of DNA fragments as a function of the relative concentration of the nuclides in the nucleus and in the cytoplasm, were also calculated. In the same conditions, the number of complex lesions (which have a high probability of inducing lethal damage to the cells) per Gy (circa 0.5-1) and the total number of double strand breaks (DSBs) per Gy (circa 40) were also calculated. To complete the characterization of the effects of internal emitters inside the cell the distributions of DSBs per chromosome were studied for different radionuclide distributions in the cell. The results obtained from these simulations show the possible overestimation or underestimation of the

Mitochondrial DNA damage is associated with damage accrual and disease duration in patients with systemic lupus erythematosus.

PubMed

López-López, L; Nieves-Plaza, M; Castro, M del R; Font, Y M; Torres-Ramos, C A; Vilá, L M; Ayala-Peña, S

2014-10-01

To determine the extent of mitochondrial DNA (mtDNA) damage in systemic lupus erythematosus (SLE) patients compared to healthy subjects and to determine the factors associated with mtDNA damage among SLE patients. A cross-sectional study was performed in 86 SLE patients (per American College of Rheumatology classification criteria) and 86 healthy individuals matched for age and gender. Peripheral blood mononuclear cells (PBMCs) were collected from subjects to assess the relative amounts of mtDNA damage. Quantitative polymerase chain reaction assay was used to measure the frequency of mtDNA lesions and mtDNA abundance. Socioeconomic-demographic features, clinical manifestations, pharmacologic treatment, disease activity, and damage accrual were determined. Statistical analyses were performed using t test, pairwise correlation, and Pearson's chi-square test (or Fisher's exact test) as appropriate. Among SLE patients, 93.0% were women. The mean (SD) age was 38.0 (10.4) years and the mean (SD) disease duration was 8.7 (7.5) years. SLE patients exhibited increased levels of mtDNA damage as shown by higher levels of mtDNA lesions and decreased mtDNA abundance as compared to healthy individuals. There was a negative correlation between disease damage and mtDNA abundance and a positive correlation between mtDNA lesions and disease duration. No association was found between disease activity and mtDNA damage. PBMCs from SLE patients exhibited more mtDNA damage compared to healthy subjects. Higher levels of mtDNA damage were observed among SLE patients with major organ involvement and damage accrual. These results suggest that mtDNA damage have a potential role in the pathogenesis of SLE. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Repair of DNA Damage Induced by the Cytidine Analog Zebularine Requires ATR and ATM in Arabidopsis[OPEN

PubMed Central

Liu, Chun-Hsin; Finke, Andreas; Díaz, Mariana; Rozhon, Wilfried; Poppenberger, Brigitte; Baubec, Tuncay; Pecinka, Ales

2015-01-01

DNA damage repair is an essential cellular mechanism that maintains genome stability. Here, we show that the nonmethylable cytidine analog zebularine induces a DNA damage response in Arabidopsis thaliana, independent of changes in DNA methylation. In contrast to genotoxic agents that induce damage in a cell cycle stage-independent manner, zebularine induces damage specifically during strand synthesis in DNA replication. The signaling of this damage is mediated by additive activity of ATAXIA TELANGIECTASIA MUTATED AND RAD3-RELATED and ATAXIA TELANGIECTASIA MUTATED kinases, which cause postreplicative cell cycle arrest and increased endoreplication. The repair requires a functional STRUCTURAL MAINTENANCE OF CHROMOSOMES5 (SMC5)-SMC6 complex and is accomplished predominantly by synthesis-dependent strand-annealing homologous recombination. Here, we provide insight into the response mechanism for coping with the genotoxic effects of zebularine and identify several components of the zebularine-induced DNA damage repair pathway. PMID:26023162

Evaluating In Vitro DNA Damage Using Comet Assay.

PubMed

Lu, Yanxin; Liu, Yang; Yang, Chunzhang

2017-10-11

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer therapies, such as radio- and chemotherapy, introduce enormous amount of additional DNA damage, leading to cell cycle arrest and apoptosis to limit cancer progression. Quantitative assessment of DNA damage during experimental cancer therapy is a key step to justify the effectiveness of a genotoxic agent. In this study, we focus on a single cell electrophoresis assay, also known as the comet assay, which can quantify single and double-strand DNA breaks in vitro. The comet assay is a DNA damage quantification method that is efficient and easy to perform, and has low time/budget demands and high reproducibility. Here, we highlight the utility of the comet assay for a preclinical study by evaluating the genotoxic effect of olaparib/temozolomide combination therapy to U251 glioma cells.

PHD3-dependent hydroxylation of HCLK2 promotes the DNA damage response

PubMed Central

Xie, Liang; Pi, Xinchun; Mishra, Ashutosh; Fong, Guohua; Peng, Junmin; Patterson, Cam

2012-01-01

The DNA damage response (DDR) is a complex regulatory network that is critical for maintaining genome integrity. Posttranslational modifications are widely used to ensure strict spatiotemporal control of signal flow, but how the DDR responds to environmental cues, such as changes in ambient oxygen tension, remains poorly understood. We found that an essential component of the ATR/CHK1 signaling pathway, the human homolog of the Caenorhabditis elegans biological clock protein CLK-2 (HCLK2), associated with and was hydroxylated by prolyl hydroxylase domain protein 3 (PHD3). HCLK2 hydroxylation was necessary for its interaction with ATR and the subsequent activation of ATR/CHK1/p53. Inhibiting PHD3, either with the pan-hydroxylase inhibitor dimethyloxaloylglycine (DMOG) or through hypoxia, prevented activation of the ATR/CHK1/p53 pathway and decreased apoptosis induced by DNA damage. Consistent with these observations, we found that mice lacking PHD3 were resistant to the effects of ionizing radiation and had decreased thymic apoptosis, a biomarker of genomic integrity. Our identification of HCLK2 as a substrate of PHD3 reveals the mechanism through which hypoxia inhibits the DDR, suggesting hydroxylation of HCLK2 is a potential therapeutic target for regulating the ATR/CHK1/p53 pathway. PMID:22797300

Ubiquitin-proteasome pathway and cellular responses to oxidative stress

USDA-ARS?s Scientific Manuscript database

The ubiquitin-proteasome pathway (UPP) is the primary cytosolic proteolytic machinery for the selective degradation of various forms of damaged proteins. Thus, the UPP is an important protein quality control mechanism. In the canonical UPP, both ubiquitin and the 26S proteasome are involved. Subs...

Eukaryotic DNA polymerase ζ

PubMed Central

Makarova, Alena V.; Burgers, Peter M.

2015-01-01

This review focuses on eukaryotic DNA polymerase ζ (Pol ζ), the enzyme responsible for the bulk of mutagenesis in eukaryotic cells in response to DNA damage. Pol ζ is also responsible for a large portion of mutagenesis during normal cell growth, in response to spontaneous damage or to certain DNA structures and other blocks that stall DNA replication forks. Novel insights in mutagenesis have been derived from recent advances in the elucidation of the subunit structure of Pol ζ. The lagging strand DNA polymerase δ shares the small Pol31 and Pol32 subunits with the Rev3-Rev7 core assembly giving a four subunit Pol ζ complex that is the active form in mutagenesis. Furthermore, Pol ζ forms essential interactions with the mutasome assembly factor Rev1 and with proliferating cell nuclear antigen (PCNA). These interactions are modulated by posttranslational modifications such as ubiquitination and phosphorylation that enhance translesion synthesis (TLS) and mutagenesis. PMID:25737057

DNA damage in cells exhibiting radiation-induced genomic instability

DOE PAGES

Keszenman, Deborah J.; Kolodiuk, Lucia; Baulch, Janet E.

2015-02-22

Cells exhibiting radiation induced genomic instability exhibit varied spectra of genetic and chromosomal aberrations. Even so, oxidative stress remains a common theme in the initiation and/or perpetuation of this phenomenon. Isolated oxidatively modified bases, abasic sites, DNA single strand breaks and clustered DNA damage are induced in normal mammalian cultured cells and tissues due to endogenous reactive oxygen species generated during normal cellular metabolism in an aerobic environment. While sparse DNA damage may be easily repaired, clustered DNA damage may lead to persistent cytotoxic or mutagenic events that can lead to genomic instability. In this study, we tested the hypothesismore » that DNA damage signatures characterised by altered levels of endogenous, potentially mutagenic, types of DNA damage and chromosomal breakage are related to radiation-induced genomic instability and persistent oxidative stress phenotypes observed in the chromosomally unstable progeny of irradiated cells. The measurement of oxypurine, oxypyrimidine and abasic site endogenous DNA damage showed differences in non-double-strand breaks (DSB) clusters among the three of the four unstable clones evaluated as compared to genomically stable clones and the parental cell line. These three unstable clones also had increased levels of DSB clusters. The results of this study demonstrate that each unstable cell line has a unique spectrum of persistent damage and lead us to speculate that alterations in DNA damage signaling and repair may be related to the perpetuation of genomic instability.« less

Structural Basis of Mec1-Ddc2-RPA Assembly and Activation on Single-Stranded DNA at Sites of Damage.

PubMed

Deshpande, Ishan; Seeber, Andrew; Shimada, Kenji; Keusch, Jeremy J; Gut, Heinz; Gasser, Susan M

2017-10-19

Mec1-Ddc2 (ATR-ATRIP) is a key DNA-damage-sensing kinase that is recruited through the single-stranded (ss) DNA-binding replication protein A (RPA) to initiate the DNA damage checkpoint response. Activation of ATR-ATRIP in the absence of DNA damage is lethal. Therefore, it is important that damage-specific recruitment precedes kinase activation, which is achieved at least in part by Mec1-Ddc2 homodimerization. Here, we report a structural, biochemical, and functional characterization of the yeast Mec1-Ddc2-RPA assembly. High-resolution co-crystal structures of Ddc2-Rfa1 and Ddc2-Rfa1-t11 (K45E mutant) N termini and of the Ddc2 coiled-coil domain (CCD) provide insight into Mec1-Ddc2 homodimerization and damage-site targeting. Based on our structural and functional findings, we present a Mec1-Ddc2-RPA-ssDNA composite structural model. By way of validation, we show that RPA-dependent recruitment of Mec1-Ddc2 is crucial for maintaining its homodimeric state at ssDNA and that Ddc2's recruitment domain and CCD are important for Mec1-dependent survival of UV-light-induced DNA damage. Copyright © 2017 Elsevier Inc. All rights reserved.

Spatiotemporal recruitment of human DNA polymerase delta to sites of UV damage

PubMed Central

Chea, Jennifer; Zhang, Sufang; Zhao, Hong; Zhang, Zhongtao; Lee, Ernest Y.C.; Darzynkiewicz, Zbigniew; Lee, Marietta Y.W.T.

2012-01-01

Human DNA polymerase δ (Pol δ) is involved in various DNA damage responses in addition to its central role in DNA replication. The Pol δ4 holoenzyme consists of four subunits, p125, p50, p68 and p12. It has been established that the p12 subunit is rapidly degraded in response to DNA damage by UV leading to the in vivo conversion of Pol δ4 to Pol δ3, a trimeric form lacking the p12 subunit. We provide the first analysis of the time-dependent recruitment of the individual Pol δ subunits to sites of DNA damage produced by UV irradiation through 5 μm polycarbonate filters by immunofluorescence microscopy and laser scanning cytometry (LSC). Quantitative analysis demonstrates that the recruitments of the three large subunits was near complete by 2 h and did not change significantly up to 4 h after UV exposure. However, the recruitment of p12 was incomplete even at 4 h, with about 70% of the Pol δ lacking the p12 subunit. ChIP analysis of Pol δ after global UV irradiation further demonstrates that only p125, p50 and p68 were present. Thus, Pol δ3 is the predominant form of Pol δ at sites of UV damage as a result of p12 degradation. Using LSC, we have further confirmed that Pol δ was recruited to CPD damage sites in all phases of the cell cycle. Collectively, our results show that Pol δ at the DNA damage site is the Pol δ trimer lacking p12 regardless of the cell cycle phase. PMID:22801543

Ubiquitin ligase parkin promotes Mdm2-arrestin interaction but inhibits arrestin ubiquitination

PubMed Central

Ahmed, M. Rafiuddin; Zhan, Xuanzhi; Song, Xiufeng; Kook, Seunghyi; Gurevich, Vsevolod V.; Gurevich, Eugenia V.

2011-01-01

Numerous mutations in E3 ubiquitin ligase parkin were shown to associate with familial Parkinson's disease. Here we show that parkin binds arrestins, versatile regulators of cell signaling. Arrestin-parkin interaction was demonstrated by coimmuno-precipitation of endogenous proteins from brain tissue, and shown to be direct using purified proteins. Parkin binding enhances arrestin interactions with another E3 ubiquitin ligase, Mdm2, apparently by shifting arrestin conformational equilibrium to the basal state preferred by Mdm2. Although Mdm2 was reported to ubiquitinate arrestins, parkin-dependent increase in Mdm2 binding dramatically reduces the ubiquitination of both non-visual arrestins, basal and stimulated by receptor activation, without affecting receptor internalization. Several disease-associated parkin mutations differentially affect the stimulation of Mdm2 binding. All parkin mutants tested effectively suppress arrestin ubiquitination, suggesting that bound parkin shields arrestin lysines targeted by Mdm2. Parkin binding to arrestins along with its effects on arrestin interaction with Mdm2 and ubiquitination is a novel function of this protein with implications for Parkinson's disease pathology. PMID:21466165

Ubiquitin ligase parkin promotes Mdm2-arrestin interaction but inhibits arrestin ubiquitination.

PubMed

Ahmed, M Rafiuddin; Zhan, Xuanzhi; Song, Xiufeng; Kook, Seunghyi; Gurevich, Vsevolod V; Gurevich, Eugenia V

2011-05-10

Numerous mutations in E3 ubiquitin ligase parkin were shown to associate with familial Parkinson's disease. Here we show that parkin binds arrestins, versatile regulators of cell signaling. Arrestin-parkin interaction was demonstrated by coimmunoprecipitation of endogenous proteins from brain tissue and shown to be direct using purified proteins. Parkin binding enhances arrestin interactions with another E3 ubiquitin ligase, Mdm2, apparently by shifting arrestin conformational equilibrium to the basal state preferred by Mdm2. Although Mdm2 was reported to ubiquitinate arrestins, parkin-dependent increase in Mdm2 binding dramatically reduces the ubiquitination of both nonvisual arrestins, basal and stimulated by receptor activation, without affecting receptor internalization. Several disease-associated parkin mutations differentially affect the stimulation of Mdm2 binding. All parkin mutants tested effectively suppress arrestin ubiquitination, suggesting that bound parkin shields arrestin lysines targeted by Mdm2. Parkin binding to arrestins along with its effects on arrestin interaction with Mdm2 and ubiquitination is a novel function of this protein with implications for Parkinson's disease pathology.

The RNA Splicing Response to DNA Damage.

PubMed

Shkreta, Lulzim; Chabot, Benoit

2015-10-29

The number of factors known to participate in the DNA damage response (DDR) has expanded considerably in recent years to include splicing and alternative splicing factors. While the binding of splicing proteins and ribonucleoprotein complexes to nascent transcripts prevents genomic instability by deterring the formation of RNA/DNA duplexes, splicing factors are also recruited to, or removed from, sites of DNA damage. The first steps of the DDR promote the post-translational modification of splicing factors to affect their localization and activity, while more downstream DDR events alter their expression. Although descriptions of molecular mechanisms remain limited, an emerging trend is that DNA damage disrupts the coupling of constitutive and alternative splicing with the transcription of genes involved in DNA repair, cell-cycle control and apoptosis. A better understanding of how changes in splice site selection are integrated into the DDR may provide new avenues to combat cancer and delay aging.

Role of oxidants in DNA damage. Hydroxyl radical mediates the synergistic DNA damaging effects of asbestos and cigarette smoke.

PubMed Central

Jackson, J H; Schraufstatter, I U; Hyslop, P A; Vosbeck, K; Sauerheber, R; Weitzman, S A; Cochrane, C G

1987-01-01

The mechanism by which cigarette smoking and asbestos exposure synergistically increase the incidence of lung cancer is unknown. We hypothesized that cigarette smoke and asbestos might synergistically increase DNA damage. To test this hypothesis we exposed isolated bacteriophage PM2 DNA to cigarette smoke and/or asbestos, and assessed DNA strand breaks as an index of DNA damage. Our results supported our hypothesis. 78 +/- 12% of the DNA exposed to both cigarette smoke and asbestos developed strand breaks, while only 9.8 +/- 7.0 or 4.3 +/- 3.3% of the DNA exposed to cigarette smoke or asbestos, respectively, developed strand breaks under the conditions of the experiment. Our experimental evidence suggested that cigarette smoke and asbestos synergistically increased DNA damage by stimulating .OH formation. First, significant amounts of .OH were detected by electron paramagnetic resonance (EPR) in DNA mixtures containing both cigarette smoke and asbestos, but no .OH was detected in mixtures containing cigarette smoke alone or asbestos alone. Second, the .OH scavengers, dimethylsulfoxide (DMSO), mannitol, or Na benzoate decreased both .OH detection by EPR and strand breaks in DNA mixtures exposed to cigarette smoke and asbestos. Third, the H2O2 scavenger, catalase, and the iron chelators, 1,10-phenanthroline and desferrithiocin, decreased both .OH detection and strand breaks in DNA mixtures exposed to cigarette smoke and asbestos. These latter findings suggest that iron contained in asbestos may catalyze the formation of .OH from H2O2 generated by cigarette smoke. In summary, our study indicates that cigarette smoke and asbestos synergistically increase DNA damage and suggests that this synergism may involve .OH production. PMID:2821073

ASCIZ regulates lesion-specific Rad51 focus formation and apoptosis after methylating DNA damage

PubMed Central

McNees, Carolyn J; Conlan, Lindus A; Tenis, Nora; Heierhorst, Jörg

2005-01-01

Nuclear Rad51 focus formation is required for homology-directed repair of DNA double-strand breaks (DSBs), but its regulation in response to non-DSB lesions is poorly understood. Here we report a novel human SQ/TQ cluster domain-containing protein termed ASCIZ that forms Rad51-containing foci in response to base-modifying DNA methylating agents but not in response to DSB-inducing agents. ASCIZ foci seem to form prior to Rad51 recruitment, and an ASCIZ core domain can concentrate Rad51 in focus-like structures independently of DNA damage. ASCIZ depletion dramatically increases apoptosis after methylating DNA damage and impairs Rad51 focus formation in response to methylating agents but not after ionizing radiation. ASCIZ focus formation and increased apoptosis in ASCIZ-depleted cells depend on the mismatch repair protein MLH1. Interestingly, ASCIZ foci form efficiently during G1 phase, when sister chromatids are unavailable as recombination templates. We propose that ASCIZ acts as a lesion-specific focus scaffold in a Rad51-dependent pathway that resolves cytotoxic repair intermediates, most likely single-stranded DNA gaps, resulting from MLH1-dependent processing of base lesions. PMID:15933716

ASCIZ regulates lesion-specific Rad51 focus formation and apoptosis after methylating DNA damage.

PubMed

McNees, Carolyn J; Conlan, Lindus A; Tenis, Nora; Heierhorst, Jörg

2005-07-06

Nuclear Rad51 focus formation is required for homology-directed repair of DNA double-strand breaks (DSBs), but its regulation in response to non-DSB lesions is poorly understood. Here we report a novel human SQ/TQ cluster domain-containing protein termed ASCIZ that forms Rad51-containing foci in response to base-modifying DNA methylating agents but not in response to DSB-inducing agents. ASCIZ foci seem to form prior to Rad51 recruitment, and an ASCIZ core domain can concentrate Rad51 in focus-like structures independently of DNA damage. ASCIZ depletion dramatically increases apoptosis after methylating DNA damage and impairs Rad51 focus formation in response to methylating agents but not after ionizing radiation. ASCIZ focus formation and increased apoptosis in ASCIZ-depleted cells depend on the mismatch repair protein MLH1. Interestingly, ASCIZ foci form efficiently during G1 phase, when sister chromatids are unavailable as recombination templates. We propose that ASCIZ acts as a lesion-specific focus scaffold in a Rad51-dependent pathway that resolves cytotoxic repair intermediates, most likely single-stranded DNA gaps, resulting from MLH1-dependent processing of base lesions.

Fisetin Protects DNA Against Oxidative Damage and Its Possible Mechanism.

PubMed

Wang, Tingting; Lin, Huajuan; Tu, Qian; Liu, Jingjing; Li, Xican

2016-06-01

The paper tries to assess the protective effect of fisetin against •OH-induced DNA damage, then to investigate the possible mechanism. The protective effect was evaluated based on the content of malondialdehyde (MDA). The possible mechanism was analyzed using various antioxidant methods in vitro, including •OH scavenging (deoxyribose degradation), •O2 (-) scavenging (pyrogallol autoxidation), DPPH• scavenging, ABTS•(+) scavenging, and Cu(2+)-reducing power assays. Fisetin increased dose-dependently its protective percentages against •OH-induced DNA damage (IC50 value =1535.00±29.60 µM). It also increased its radical-scavenging percentages in a dose-dependent manner in various antioxidants assays. Its IC50 values in •OH scavenging, •O2(-) scavenging, DPPH• scavenging, ABTS•(+) scavenging, and Cu(2+)-reducing power assays, were 47.41±4.50 µM, 34.05±0.87 µM, 9.69±0.53 µM, 2.43±0.14 µM, and 1.49±0.16 µM, respectively. Fisetin can effectively protect DNA against •OH-induced oxidative damage possibly via reactive oxygen species (ROS) scavenging approach, which is assumed to be hydrogen atom (H•) and/or single electron (e) donation (HAT/SET) pathways. In the HAT pathway, the 3',4'-dihydroxyl moiety in B ring of fisetin is thought to play an important role, because it can be ultimately oxidized to a stable ortho-benzoquinone form.

Fisetin Protects DNA Against Oxidative Damage and Its Possible Mechanism

PubMed Central

Wang, Tingting; Lin, Huajuan; Tu, Qian; Liu, Jingjing; Li, Xican

2016-01-01

Purpose: The paper tries to assess the protective effect of fisetin against •OH-induced DNA damage, then to investigate the possible mechanism. Methods: The protective effect was evaluated based on the content of malondialdehyde (MDA). The possible mechanism was analyzed using various antioxidant methods in vitro, including •OH scavenging (deoxyribose degradation), •O2- scavenging (pyrogallol autoxidation), DPPH• scavenging, ABTS•+ scavenging, and Cu2+-reducing power assays. Results: Fisetin increased dose-dependently its protective percentages against •OH-induced DNA damage (IC50 value =1535.00±29.60 µM). It also increased its radical-scavenging percentages in a dose-dependent manner in various antioxidants assays. Its IC50 values in •OH scavenging, •O2- scavenging, DPPH• scavenging, ABTS•+ scavenging, and Cu2+-reducing power assays, were 47.41±4.50 µM, 34.05±0.87 µM, 9.69±0.53 µM, 2.43±0.14 µM, and 1.49±0.16 µM, respectively. Conclusion: Fisetin can effectively protect DNA against •OH-induced oxidative damage possibly via reactive oxygen species (ROS) scavenging approach, which is assumed to be hydrogen atom (H•) and/or single electron (e) donation (HAT/SET) pathways. In the HAT pathway, the 3’,4’-dihydroxyl moiety in B ring of fisetin is thought to play an important role, because it can be ultimately oxidized to a stable ortho-benzoquinone form. PMID:27478791

Protoparvovirus Interactions with the Cellular DNA Damage Response

PubMed Central

Majumder, Kinjal; Etingov, Igor

2017-01-01

Protoparvoviruses are simple single-stranded DNA viruses that infect many animal species. The protoparvovirus minute virus of mice (MVM) infects murine and transformed human cells provoking a sustained DNA damage response (DDR). This DDR is dependent on signaling by the ATM kinase and leads to a prolonged pre-mitotic cell cycle block that features the inactivation of ATR-kinase mediated signaling, proteasome-targeted degradation of p21, and inhibition of cyclin B1 expression. This review explores how protoparvoviruses, and specifically MVM, co-opt the common mechanisms regulating the DDR and cell cycle progression in order to prepare the host nuclear environment for productive infection. PMID:29088070

Protoparvovirus Interactions with the Cellular DNA Damage Response.

PubMed

Majumder, Kinjal; Etingov, Igor; Pintel, David J

2017-10-31

Protoparvoviruses are simple single-stranded DNA viruses that infect many animal species. The protoparvovirus minute virus of mice (MVM) infects murine and transformed human cells provoking a sustained DNA damage response (DDR). This DDR is dependent on signaling by the ATM kinase and leads to a prolonged pre-mitotic cell cycle block that features the inactivation of ATR-kinase mediated signaling, proteasome-targeted degradation of p21, and inhibition of cyclin B1 expression. This review explores how protoparvoviruses, and specifically MVM, co-opt the common mechanisms regulating the DDR and cell cycle progression in order to prepare the host nuclear environment for productive infection.

Metabolic responses induced by DNA damage and poly (ADP-ribose) polymerase (PARP) inhibition in MCF-7 cells

PubMed Central

Bhute, Vijesh J.; Palecek, Sean P.

2015-01-01

Genomic instability is one of the hallmarks of cancer. Several chemotherapeutic drugs and radiotherapy induce DNA damage to prevent cancer cell replication. Cells in turn activate different DNA damage response (DDR) pathways to either repair the damage or induce cell death. These DDR pathways also elicit metabolic alterations which can play a significant role in the proper functioning of the cells. The understanding of these metabolic effects resulting from different types of DNA damage and repair mechanisms is currently lacking. In this study, we used NMR metabolomics to identify metabolic pathways which are altered in response to different DNA damaging agents. By comparing the metabolic responses in MCF-7 cells, we identified the activation of poly (ADP-ribose) polymerase (PARP) in methyl methanesulfonate (MMS)-induced DNA damage. PARP activation led to a significant depletion of NAD+. PARP inhibition using veliparib (ABT-888) was able to successfully restore the NAD+ levels in MMS-treated cells. In addition, double strand break induction by MMS and veliparib exhibited similar metabolic responses as zeocin, suggesting an application of metabolomics to classify the types of DNA damage responses. This prediction was validated by studying the metabolic responses elicited by radiation. Our findings indicate that cancer cell metabolic responses depend on the type of DNA damage responses and can also be used to classify the type of DNA damage. PMID:26478723

Proteostasis regulation by the ubiquitin system.

PubMed

Bett, John S

2016-10-15

Cells have developed an evolutionary obligation to survey and maintain proteome fidelity and avoid the possible toxic consequences of protein misfolding and aggregation. Disturbances to protein homoeostasis (proteostasis) can result in severe cellular phenotypes and are closely linked with the accumulation of microscopically visible deposits of aggregated proteins. These include inclusion bodies found in AD (Alzheimer's disease), HD (Huntington's disease) and ALS (amyotrophic lateral sclerosis) patient neurons. Protein aggregation is intimately linked with the ubiquitin and ubiquitin-like post-translational modifier system, which manages cellular protein folding stress and promotes the restoration of proteostasis. This is achieved in large part through the action of the UPS (ubiquitin-proteasome system), which is responsible for directing the proteasomal destruction of misfolded and damaged proteins tagged with ubiquitin chains. There are other less well understood ways in which ubiquitin family members can help to maintain proteostasis that complement, but are independent of, the UPS. This article discusses our current understanding of how the ubiquitin family regulates the protein misfolding pathways that threaten proteome fidelity, and how this is achieved by the key players in this process. © 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

PINK1 autophosphorylation is required for ubiquitin recognition.

PubMed

Rasool, Shafqat; Soya, Naoto; Truong, Luc; Croteau, Nathalie; Lukacs, Gergely L; Trempe, Jean-François

2018-04-01

Mutations in PINK1 cause autosomal recessive Parkinson's disease (PD), a neurodegenerative movement disorder. PINK1 is a kinase that acts as a sensor of mitochondrial damage and initiates Parkin-mediated clearance of the damaged organelle. PINK1 phosphorylates Ser65 in both ubiquitin and the ubiquitin-like (Ubl) domain of Parkin, which stimulates its E3 ligase activity. Autophosphorylation of PINK1 is required for Parkin activation, but how this modulates the ubiquitin kinase activity is unclear. Here, we show that autophosphorylation of Tribolium castaneum PINK1 is required for substrate recognition. Using enzyme kinetics and NMR spectroscopy, we reveal that PINK1 binds the Parkin Ubl with a 10-fold higher affinity than ubiquitin via a conserved interface that is also implicated in RING1 and SH3 binding. The interaction requires phosphorylation at Ser205, an invariant PINK1 residue (Ser228 in human). Using mass spectrometry, we demonstrate that PINK1 rapidly autophosphorylates in trans at Ser205. Small-angle X-ray scattering and hydrogen-deuterium exchange experiments provide insights into the structure of the PINK1 catalytic domain. Our findings suggest that multiple PINK1 molecules autophosphorylate first prior to binding and phosphorylating ubiquitin and Parkin. © 2018 The Authors.

Parvovirus infection-induced DNA damage response

PubMed Central

Luo, Yong; Qiu, Jianming

2014-01-01

Parvoviruses are a group of small DNA viruses with ssDNA genomes flanked by two inverted terminal structures. Due to a limited genetic resource they require host cellular factors and sometimes a helper virus for efficient viral replication. Recent studies have shown that parvoviruses interact with the DNA damage machinery, which has a significant impact on the life cycle of the virus as well as the fate of infected cells. In addition, due to special DNA structures of the viral genomes, parvoviruses are useful tools for the study of the molecular mechanisms underlying viral infection-induced DNA damage response (DDR). This review aims to summarize recent advances in parvovirus-induced DDR, with a focus on the diverse DDR pathways triggered by different parvoviruses and the consequences of DDR on the viral life cycle as well as the fate of infected cells. PMID:25429305

M-phase kinases induce phospho-dependent ubiquitination of somatic Wee1 by SCFβ-TrCP

PubMed Central

Watanabe, Nobumoto; Arai, Harumi; Nishihara, Yoshifumi; Taniguchi, Makoto; Watanabe, Naoko; Hunter, Tony; Osada, Hiroyuki

2004-01-01

Wee1, the Cdc2 inhibitory kinase, needs to be down-regulated at the onset of mitosis to ensure rapid activation of Cdc2. Previously, we have shown that human somatic Wee1 (Wee1A) is down-regulated both by protein phosphorylation and degradation, but the underlying mechanisms had not been elucidated. In the present study, we have identified the β-transducin repeat-containing protein 1/2 (β-TrCP1/2) F-box protein-containing SKP1/Cul1/F-box protein (SCF) complex (SCFβ-TrCP1/2) as an E3 ubiquitin ligase for Wee1A ubiquitination. Although Wee1A lacks a consensus DS(p)GXXS(p) phospho-dependent binding motif for β-TrCP, recognition of Wee1A by β-TrCP depended on phosphorylation, and two serine residues in Wee1A, S53 and S123, were found to be the most important phosphorylation sites for β-TrCP recognition. We have found also that the major M-phase kinases polo-like kinase 1 (Plk1) and Cdc2 are responsible for the phosphorylation of S53 and S123, respectively, and that in each case phosphorylation generates an unconventional phospho-degron (signal for degradation) that can be recognized by β-TrCP. Phosphorylation of Wee1A by these kinases cooperatively stimulated the recognition and ubiquitination of Wee1A by SCFβ-TrCP1/2 in vitro. Mutation of these residues or depletion of β-TrCP by small-interfering RNA treatment increased the stability of Wee1A in HeLa cells. Moreover, our analysis indicates that β-TrCP-dependent degradation of Wee1A is important for the normal onset of M-phase in vivo. These results also establish the existence of a feedback loop between Cdc2 and Wee1A in somatic cells that depends on ubiquitination and protein degradation and ensures the rapid activation of Cdc2 when cells are ready to divide. PMID:15070733

Ultrasound Imaging of DNA-Damage Effects in Live Cultured Cells and in Brain Tissue.

PubMed

Tadayyon, Hadi; Gangeh, Mehrdad J; Vlad, Roxana; Kolios, Michael C; Czarnota, Gregory J

2017-01-01

High-frequency ultrasound (>20 MHz) spectroscopy can be used to detect noninvasively DNA damage in cell samples in vitro, and in live tissue both ex vivo and in vivo. This chapter focuses on the former two aspects. Experimental evidence suggests that morphological changes that occur in cells undergoing apoptosis result in changes in frequency-dependent ultrasound backscatter. With advances in research, ultrasound spectroscopy is advancing the boundaries of fast, label-free, noninvasive DNA damage detection technology with potential use in personalized medicine and early therapy response monitoring. Depending on the desired resolution, parametric ultrasound images can be computed and displayed within minutes to hours after ultrasound examination for cell death.

Phospho-ubiquitin-PARK2 complex as a marker for mitophagy defects.

PubMed

Callegari, Sylvie; Oeljeklaus, Silke; Warscheid, Bettina; Dennerlein, Sven; Thumm, Michael; Rehling, Peter; Dudek, Jan

2017-01-02

The E3 ubiquitin ligase PARK2 and the mitochondrial protein kinase PINK1 are required for the initiation of mitochondrial damage-induced mitophagy. Together, PARK2 and PINK1 generate a phospho-ubiquitin signal on outer mitochondrial membrane proteins that triggers recruitment of the autophagy machinery. This paper describes the detection of a defined 500-kDa phospho-ubiquitin-rich PARK2 complex that accumulates on mitochondria upon treatment with the membrane uncoupler CCCP. Formation of this complex is dependent on the presence of PINK1 and is absent in mutant forms of PARK2, whereby mitophagy is also arrested. These results signify a functional signaling complex that is essential for the progression of mitophagy. The visualization of the PARK2 signaling complex represents a novel marker for this critical step in mitophagy and can be used to monitor mitophagy progression in PARK2 mutants and to uncover additional upstream factors required for PARK2-mediated mitophagy signaling.

Bimodal regulation of p21waf1 protein as function of DNA damage levels

PubMed Central

Buscemi, G; Ricci, C; Zannini, L; Fontanella, E; Plevani, P; Delia, D

2014-01-01

Human p21Waf1 protein is well known for being transcriptionally induced by p53 and activating the cell cycle checkpoint arrest in response to DNA breaks. Here we report that p21Waf1 protein undergoes a bimodal regulation, being upregulated in response to low doses of DNA damage but rapidly and transiently degraded in response to high doses of DNA lesions. Responsible for this degradation is the checkpoint kinase Chk1, which phosphorylates p21Waf1 on T145 and S146 residues and induces its proteasome-dependent proteolysis. The initial p21Waf1 degradation is then counteracted by the ATM-Chk2 pathway, which promotes the p53-dependent accumulation of p21Waf1 at any dose of damage. We also found that p21Waf1 ablation favors the activation of an apoptotic program to eliminate otherwise irreparable cells. These findings support a model in which in human cells a balance between ATM-Chk2-p53 and the ATR-Chk1 pathways modulates p21Waf1 protein levels in relation to cytostatic and cytotoxic doses of DNA damage. PMID:25486478

The mechanism of OTUB1-mediated inhibition of ubiquitination

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wiener, Reuven; Zhang, Xiangbin; Wang, Tao

2013-04-08