scholarly journals Multiple classes of bactericidal antibiotics cause DNA double strand breaks in Staphylococcus aureus

2021 ◽  
Author(s):  
Rebecca S. Clarke ◽  
Kam Pou Ha ◽  
Andrew M. Edwards
Keyword(s):  
Staphylococcus Aureus ◽  
Dna Damage ◽  
Double Strand Breaks ◽  
Bacterial Survival ◽  
Dna Double Strand Breaks ◽  
Anaerobic Conditions ◽  
Strand Breaks ◽  
Endogenous Production ◽  
Dna Dsbs ◽  
Dose Dependent

AbstractAntibiotics inhibit essential bacterial processes, resulting in arrest of growth and in some cases cell death. Many antibiotics are also reported to trigger endogenous production of reactive oxygen species (ROS), which damage DNA and other macromolecules. However, the type of DNA damage that arises and the mechanisms used by bacteria to repair it are largely unclear. We found that several different classes of antibiotic triggered dose-dependent DNA damage in Staphylococcus aureus, including some bacteriostatic drugs. Damage was heterogenous and varied in magnitude between strains. However, antibiotic-triggered DNA damage led to double strand breaks, the processing of which by the RexAB helicase/nuclease complex triggered the SOS response and reduced staphylococcal susceptibility to most of the antibacterials tested. In most cases, DNA DSBs occurred under aerobic but not anaerobic conditions, suggesting a role for ROS. We conclude that DNA double strand breaks are a common occurrence during bacterial exposure to several different antibiotic classes and that repair of this damage by the RexAB complex promotes bacterial survival.

scholarly journals RexAB promotes the survival of Staphylococcus aureus exposed to multiple classes of antibiotics

2021 ◽  
Author(s):  
Rebecca S. Clarke ◽  
Kam Pou Ha ◽  
Andrew M. Edwards
Keyword(s):  
Reactive Oxygen Species ◽  
Staphylococcus Aureus ◽  
Dna Damage ◽  
Sos Response ◽  
Double Strand Breaks ◽  
Oxygen Species ◽  
Bacterial Survival ◽  
Wild Type ◽  
Dna Double Strand Breaks ◽  
Strand Breaks

Antibiotics inhibit essential bacterial processes, resulting in arrest of growth and in some cases cell death. Many antibiotics are also reported to trigger endogenous production of reactive oxygen species (ROS), which damage DNA, leading to induction of the mutagenic SOS response associated with the emergence of drug resistance. However, the type of DNA damage that arises and how this triggers the SOS response is largely unclear. We found that several different classes of antibiotic triggered dose-dependent induction of the SOS response in Staphylococcus aureus , indicative of DNA damage, including some bacteriostatic drugs. The SOS response was heterogenous and varied in magnitude between strains and antibiotics. However, in many cases, full induction of the SOS response was dependent upon the RexAB helicase/nuclease complex, which processes DNA double strand breaks to produce single-stranded DNA and facilitate RecA nucleoprotein filament formation. The importance of RexAB in repair of DNA was confirmed by measuring bacterial survival during antibiotic exposure, with most drugs having significantly greater bactericidal activity against rexB mutants relative to wild type strains. For some, but not all antibiotics there was no difference in bactericidal activity between wild type and rexB mutant under anaerobic conditions, indicative of a role for reactive oxygen species in mediating DNA damage. Taken together, this work confirms previous observations that several classes of antibiotics cause DNA damage in S. aureus and extends them by showing that processing of DNA double strand breaks by RexAB is a major trigger of the mutagenic SOS response and promotes bacterial survival.

scholarly journals The chromatin remodeler p400 ATPase facilitates Rad51-mediated repair of DNA double-strand breaks

2012 ◽  
Vol 199 (7) ◽  
pp. 1067-1081 ◽  
Author(s):  
Céline Courilleau ◽  
Catherine Chailleux ◽  
Alain Jauneau ◽  
Fanny Grimal ◽  
Sébastien Briois ◽  
...  
Keyword(s):  
Dna Damage ◽  
Chromatin Remodeling ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Dna Damage Signaling ◽  
Dna Dsbs ◽  
Dependent Processes

DNA damage signaling and repair take place in a chromatin context. Consequently, chromatin-modifying enzymes, including adenosine triphosphate–dependent chromatin remodeling enzymes, play an important role in the management of DNA double-strand breaks (DSBs). Here, we show that the p400 ATPase is required for DNA repair by homologous recombination (HR). Indeed, although p400 is not required for DNA damage signaling, DNA DSB repair is defective in the absence of p400. We demonstrate that p400 is important for HR-dependent processes, such as recruitment of Rad51 to DSB (a key component of HR), homology-directed repair, and survival after DNA damage. Strikingly, p400 and Rad51 are present in the same complex and both favor chromatin remodeling around DSBs. Altogether, our data provide a direct molecular link between Rad51 and a chromatin remodeling enzyme involved in chromatin decompaction around DNA DSBs.

scholarly journals Checkpoint Responses to DNA Double-Strand Breaks

2020 ◽  
Vol 89 (1) ◽  
pp. 103-133 ◽  
Author(s):  
David P. Waterman ◽  
James E. Haber ◽  
Marcus B. Smolka
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Dna Damage ◽  
Model System ◽  
Double Strand Breaks ◽  
Dna Damage Checkpoint ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Dna Dsbs ◽  
Cell Lethality

Cells confront DNA damage in every cell cycle. Among the most deleterious types of DNA damage are DNA double-strand breaks (DSBs), which can cause cell lethality if unrepaired or cancers if improperly repaired. In response to DNA DSBs, cells activate a complex DNA damage checkpoint (DDC) response that arrests the cell cycle, reprograms gene expression, and mobilizes DNA repair factors to prevent the inheritance of unrepaired and broken chromosomes. Here we examine the DDC, induced by DNA DSBs, in the budding yeast model system and in mammals.

scholarly journals Physical interaction between the histone acetyl transferase Tip60 and the DNA double-strand breaks sensor MRN complex

2010 ◽  
Vol 426 (3) ◽  
pp. 365-371 ◽  
Author(s):  
Catherine Chailleux ◽  
Sandrine Tyteca ◽  
Christophe Papin ◽  
François Boudsocq ◽  
Nadine Puget ◽  
...  
Keyword(s):  
Dna Damage ◽  
Mammalian Cells ◽  
Double Strand Breaks ◽  
Physical Interaction ◽  
Dna Double Strand Breaks ◽  
Reporter System ◽  
Histone Acetyl Transferase ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Dna Dsbs

Chromatin modifications and chromatin-modifying enzymes are believed to play a major role in the process of DNA repair. The histone acetyl transferase Tip60 is physically recruited to DNA DSBs (double-strand breaks) where it mediates histone acetylation. In the present study, we show, using a reporter system in mammalian cells, that Tip60 expression is required for homology-driven repair, strongly suggesting that Tip60 participates in DNA DSB repair through homologous recombination. Moreover, Tip60 depletion inhibits the formation of Rad50 foci following ionizing radiation, indicating that Tip60 expression is necessary for the recruitment of the DNA damage sensor MRN (Mre11–Rad50–Nbs1) complex to DNA DSBs. Moreover, we found that endogenous Tip60 physically interacts with endogenous MRN proteins in a complex which is distinct from the classical Tip60 complex. Taken together, our results describe a physical link between a DNA damage sensor and a histone-modifying enzyme, and provide important new insights into the role and mechanism of action of Tip60 in the process of DNA DSB repair.

scholarly journals Μελέτη των ποιοτικών και ποσοτικών χαρακτηριστικών της φλεγμονώδους αντίδρασης σε αδενοκαρκινώματα παχέος εντέρου

2018 ◽  
Author(s):  
Θεόδωρος Αργυρόπουλος
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Dna Dsbs ◽  
Damage Response

Τα κακοήθη επιθηλιακά νεοπλάσματα του παχέος εντέρου και του ορθού αποτελούν την τρίτη συχνότερη μορφή κακοήθους νεοπλασίας ενώ είναι συνολικά η δεύτερη συχνότερη αιτία θανάτου από καρκίνο μετά τον καρκίνο του πνεύμονα. Παρά την σημαντική πρόοδο στον τομέα της προεγχειρητικής απεικόνισης, των χειρουργικών τεχνικών και της χημειοθεραπείας, περίπου το 40-50% των ασθενών παρουσιάζει υποτροπή η οποία ακολουθεί μία δυνητικά θεραπευτική παρέμβαση, υποδεικνύοντας την ανάγκη για καλύτερη σταδιοποίηση και θεραπεία.Παρότι η παρουσία λευκοκυττάρων μεταξύ των νεοπλασματικών κυττάρων έχει ήδη παρατηρηθεί από τον 19ο αιώνα, οι μοριακοί μηχανισμοί, μέσω των οποίων η φλεγμονή προάγει την ανάπτυξη του καρκίνου, είναι εν πολλοίς άγνωστοι και τα διαθέσιμα μέχρι στιγμής δεδομένα λίγα. Ο ρόλος της επίκτητης ανοσολογικής απάντησης στον έλεγχο της ανάπτυξης και της επανεμφάνισης των κακοήθων νεοπλασμάτων του ανθρώπου είναι, σε πολλές περιπτώσεις, αντιφατικός. Έχει παρατηρηθεί ότι συχνά τα κακοήθη νεοπλάσματα διηθούνται από ποικίλο αριθμό λεμφοκυττάρων, μακροφάγων ή σιτευτικών κυττάρων (μαστοκυττάρων), τα οποία σχετίζονται, σε ορισμένες μελέτες, με την πρόγνωση του καρκίνου και την πενταετή επιβίωση του ασθενούς.. Συγχρόνως, άλλες παράμετροι προστίθενται διαρκώς στους υπό μελέτη πιθανούς προγνωστικούς δείκτες. Η γενετική αστάθεια αποτελεί σταθερό χαρακτηριστικό των επιθηλιακών νεοπλασμάτων του παχέος εντέρου και του ορθού, υπό μορφή είτε της χρωμοσωμιακής, είτε της μικροδορυφορικής αστάθειας. Πρόσφατα έχει καταδειχθεί ότι, σε πρώιμες βλάβες που σχετίζονται με καρκινογένεση, υπάρχει ενεργοποίηση των μηχανισμών αναγνώρισης και επιδιόρθωσης των βλαβών του DNA και κυρίως της θραύσης και των δύο ελίκων του DNA (double strand breaks), η οποία αποτελεί την πλέον επικίνδυνη μορφή βλάβης του DNA, σχετιζόμενη με γονιδιακή αστάθεια. Παρόλο που οι μοριακοί μηχανισμοί της χρωμοσωμικής αστάθειας παραμένουν στο μεγαλύτερο μέρος τους άγνωστοι, η δημιουργία θραύσεων στη διπλή έλικα του DNA (DSBs) αποτελεί έναν από αυτούς. Τα φυσιολογικά κύτταρα έχουν τη δυνατότητα να ενεργοποιήσουν έναν πολύ καλά οργανωμένο και αποτελεσματικό μηχανισμό (DNA damage response, DDR), ο οποίος αναγνωρίζει και επιδιορθώνει τις βλάβες προκειμένου να διατηρηθεί η γονιδιακή ακεραιότητα του κυττάρου. Ο μηχανισμός αυτός έχει δειχθεί ότι ενεργοποιείται σε προκαρκινωματώδεις καταστάσεις, αποτελώντας φραγμό στη διαδικασία καρκινογένεσης.Σκοπός της παρούσας μελέτης ήταν η μελέτη των ποιοτικών και ποσοτικών χαρακτηριστικών της φλεγμονώδους αντίδρασης σε αδενοκαρκινώματα του παχέος εντέρου και η συσχέτιση των ανωτέρω με το επίπεδο ενεργοποίησης των μηχανισμών απόκρισης σε βλάβες του DNA στα νεοπλασματικά κύτταρα. Τα CD3, CD8 και CD45RO φάνηκε να έχουν προγνωστική αξία στην πενταετή επιβίωση, η χρήση δε των μετρήσεων για την εκπαίδευση ενός ANN επέτρεψε την πρόβλεψη με 73% ευαισθησία και 69% ειδικότητα, με Θετική Προγνωστική Αξία στο 70%, Αρνητική Προγνωστική του Αξία στο 72%, Ψευδώς Θετικό Ποσοστό στο 31%, Ψευδώς Αρνητικό Ποσοστό στο 27%, με Συνολική Ακρίβεια στο 71%. Από το σύνολο των δεικτών που μελετήθηκαν, οι δείκτες που σχετίζονταν με τους μηχανισμούς επιδιόρθωσης (MLH-1, MSH-2, MSH-6, PMS-2, p16) και Απόκρισης στις βλάβες του DNA (γ H2AX) δεν σχετίστηκαν με την επιβίωση και περαιτέρω μελέτη απαιτείται για την κατανόηση των πολύπλοκων σχέσεων που δημιουργούνται μεταξύ των βλαβών του DNA, των μηχανισμών επιδιόρθωσης και απόκρισης και της επαγόμενης φλεγμονώδους αντίδρασης. Τέλος, προτείναμε ένα απλό, χαμηλού κόστους και εύκολα αναπαραγώγιμο σύστημα εξατομικευμένης πρόγνωσης των ασθενών με καρκίνο παχέος εντέρου σταδίου ΙΙ και ΙΙΙ.

scholarly journals PARP1 exhibits enhanced association and catalytic efficiency with γH2A.X-nucleosome

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Deepti Sharma ◽  
Louis De Falco ◽  
Sivaraman Padavattan ◽  
Chang Rao ◽  
Susana Geifman-Shochat ◽  
...  
Keyword(s):  
Dna Damage ◽  
Mutational Analysis ◽  
Catalytic Efficiency ◽  
Double Strand Breaks ◽  
Genomic Integrity ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Nucleosome Core ◽  
Epigenetic Marker ◽  
Kinetics Of

AbstractThe poly(ADP-ribose) polymerase, PARP1, plays a key role in maintaining genomic integrity by detecting DNA damage and mediating repair. γH2A.X is the primary histone marker for DNA double-strand breaks and PARP1 localizes to H2A.X-enriched chromatin damage sites, but the basis for this association is not clear. We characterize the kinetics of PARP1 binding to a variety of nucleosomes harbouring DNA double-strand breaks, which reveal that PARP1 associates faster with (γ)H2A.X- versus H2A-nucleosomes, resulting in a higher affinity for the former, which is maximal for γH2A.X-nucleosome that is also the activator eliciting the greatest poly-ADP-ribosylation catalytic efficiency. The enhanced activities with γH2A.X-nucleosome coincide with increased accessibility of the DNA termini resulting from the H2A.X-Ser139 phosphorylation. Indeed, H2A- and (γ)H2A.X-nucleosomes have distinct stability characteristics, which are rationalized by mutational analysis and (γ)H2A.X-nucleosome core crystal structures. This suggests that the γH2A.X epigenetic marker directly facilitates DNA repair by stabilizing PARP1 association and promoting catalysis.

scholarly journals RepID-deficient cancer cells are sensitized to a drug targeting p97/VCP segregase

2021 ◽  
Author(s):  
Sang-Min Jang ◽  
Christophe E. Redon ◽  
Haiqing Fu ◽  
Fred E. Indig ◽  
Mirit I. Aladjem
Keyword(s):  
Dna Damage ◽  
Cancer Cells ◽  
Cell Sorting ◽  
Clonogenic Survival ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Ubiquitinated Proteins ◽  
Damage Response ◽  
Survival Assay

Abstract Background The p97/valosin-containing protein (VCP) complex is a crucial factor for the segregation of ubiquitinated proteins in the DNA damage response and repair pathway. Objective We investigated whether blocking the p97/VCP function can inhibit the proliferation of RepID-deficient cancer cells using immunofluorescence, clonogenic survival assay, fluorescence-activated cell sorting, and immunoblotting. Result p97/VCP was recruited to chromatin and colocalized with DNA double-strand breaks in RepID-deficient cancer cells that undergo spontaneous DNA damage. Inhibition of p97/VCP induced death of RepID-depleted cancer cells. This study highlights the potential of targeting p97/VCP complex as an anticancer therapeutic approach. Conclusion Our results show that RepID is required to prevent excessive DNA damage at the endogenous levels. Localization of p97/VCP to DSB sites was induced based on spontaneous DNA damage in RepID-depleted cancer cells. Anticancer drugs targeting p97/VCP may be highly potent in RepID-deficient cells. Therefore, we suggest that p97/VCP inhibitors synergize with RepID depletion to kill cancer cells.

scholarly journals Focused Ion Microbeam Irradiation Induces Clustering of DNA Double-Strand Breaks in Heterochromatin Visualized by Nanoscale-Resolution Electron Microscopy

2021 ◽  
Vol 22 (14) ◽  
pp. 7638
Author(s):  
Yvonne Lorat ◽  
Judith Reindl ◽  
Anna Isermann ◽  
Christian Rübe ◽  
Anna A. Friedl ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Dna Damage ◽  
Spatial Clustering ◽  
Dna Lesions ◽  
Double Strand Breaks ◽  
Stimulated Emission ◽  
Nuclear Chromatin ◽  
Carbon Ions ◽  
Dna Double Strand Breaks ◽  
Strand Breaks

Background: Charged-particle radiotherapy is an emerging treatment modality for radioresistant tumors. The enhanced effectiveness of high-energy particles (such as heavy ions) has been related to the spatial clustering of DNA lesions due to highly localized energy deposition. Here, DNA damage patterns induced by single and multiple carbon ions were analyzed in the nuclear chromatin environment by different high-resolution microscopy approaches. Material and Methods: Using the heavy-ion microbeam SNAKE, fibroblast monolayers were irradiated with defined numbers of carbon ions (1/10/100 ions per pulse, ipp) focused to micrometer-sized stripes or spots. Radiation-induced lesions were visualized as DNA damage foci (γH2AX, 53BP1) by conventional fluorescence and stimulated emission depletion (STED) microscopy. At micro- and nanoscale level, DNA double-strand breaks (DSBs) were visualized within their chromatin context by labeling the Ku heterodimer. Single and clustered pKu70-labeled DSBs were quantified in euchromatic and heterochromatic regions at 0.1 h, 5 h and 24 h post-IR by transmission electron microscopy (TEM). Results: Increasing numbers of carbon ions per beam spot enhanced spatial clustering of DNA lesions and increased damage complexity with two or more DSBs in close proximity. This effect was detectable in euchromatin, but was much more pronounced in heterochromatin. Analyzing the dynamics of damage processing, our findings indicate that euchromatic DSBs were processed efficiently and repaired in a timely manner. In heterochromatin, by contrast, the number of clustered DSBs continuously increased further over the first hours following IR exposure, indicating the challenging task for the cell to process highly clustered DSBs appropriately. Conclusion: Increasing numbers of carbon ions applied to sub-nuclear chromatin regions enhanced the spatial clustering of DSBs and increased damage complexity, this being more pronounced in heterochromatic regions. Inefficient processing of clustered DSBs may explain the enhanced therapeutic efficacy of particle-based radiotherapy in cancer treatment.

scholarly journals Human papillomavirus E7 oncoprotein targets RNF168 to hijack the host DNA damage response

2019 ◽  
Vol 116 (39) ◽  
pp. 19552-19562 ◽  
Author(s):  
Justine Sitz ◽  
Sophie Anne Blanchet ◽  
Steven F. Gameiro ◽  
Elise Biquand ◽  
Tia M. Morgan ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Dna Damage ◽  
Cancer Cells ◽  
Genomic Instability ◽  
E3 Ligase ◽  
Human Papillomaviruses ◽  
Double Strand Breaks ◽  
Nuclear Bodies ◽  
Dna Double Strand Breaks ◽  
Strand Breaks

High-risk human papillomaviruses (HR-HPVs) promote cervical cancer as well as a subset of anogenital and head and neck cancers. Due to their limited coding capacity, HPVs hijack the host cell’s DNA replication and repair machineries to replicate their own genomes. How this host–pathogen interaction contributes to genomic instability is unknown. Here, we report that HPV-infected cancer cells express high levels of RNF168, an E3 ubiquitin ligase that is critical for proper DNA repair following DNA double-strand breaks, and accumulate high numbers of 53BP1 nuclear bodies, a marker of genomic instability induced by replication stress. We describe a mechanism by which HPV E7 subverts the function of RNF168 at DNA double-strand breaks, providing a rationale for increased homology-directed recombination in E6/E7-expressing cervical cancer cells. By targeting a new regulatory domain of RNF168, E7 binds directly to the E3 ligase without affecting its enzymatic activity. As RNF168 knockdown impairs viral genome amplification in differentiated keratinocytes, we propose that E7 hijacks the E3 ligase to promote the viral replicative cycle. This study reveals a mechanism by which tumor viruses reshape the cellular response to DNA damage by manipulating RNF168-dependent ubiquitin signaling. Importantly, our findings reveal a pathway by which HPV may promote the genomic instability that drives oncogenesis.

scholarly journals Focused ultrasound radiosensitizes human cancer cells by enhancement of DNA damage

2021 ◽  
Author(s):  
Xinrui Zhang ◽  
Mariana Bobeica ◽  
Michael Unger ◽  
Anastasia Bednarz ◽  
Bjoern Gerold ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Dna Damage ◽  
Cancer Cells ◽  
Metabolic Activity ◽  
Focused Ultrasound ◽  
Double Strand Breaks ◽  
High Intensity Focused Ultrasound ◽  
Dna Double Strand Breaks ◽  
Strand Breaks

Abstract Purpose High-intensity focused ultrasound (HIFU/FUS) has expanded as a noninvasive quantifiable option for hyperthermia (HT). HT in a temperature range of 40–47 °C (thermal dose CEM43 ≥ 25) could work as a sensitizer to radiation therapy (RT). Here, we attempted to understand the tumor radiosensitization effect at the cellular level after a combination treatment of FUS+RT. Methods An in vitro FUS system was developed to induce HT at frequencies of 1.147 and 1.467 MHz. Human head and neck cancer (FaDU), glioblastoma (T98G), and prostate cancer (PC-3) cells were exposed to FUS in ultrasound-penetrable 96-well plates followed by single-dose X‑ray irradiation (10 Gy). Radiosensitizing effects of FUS were investigated by cell metabolic activity (WST‑1 assay), apoptosis (annexin V assay, sub-G1 assay), cell cycle phases (propidium iodide staining), and DNA double-strand breaks (γH2A.X assay). Results The FUS intensities of 213 (1.147 MHz) and 225 W/cm2 (1.467 MHz) induced HT for 30 min at mean temperatures of 45.20 ± 2.29 °C (CEM43 = 436 ± 88) and 45.59 ± 1.65 °C (CEM43 = 447 ± 79), respectively. FUS improves the effect of RT significantly by reducing metabolic activity in T98G cells 48 h (RT: 96.47 ± 8.29%; FUS+RT: 79.38 ± 14.93%; p = 0.012) and in PC-3 cells 72 h (54.20 ± 10.85%; 41.01 ± 11.17%; p = 0.016) after therapy, but not in FaDu cells. Mechanistically, FUS+RT leads to increased apoptosis and enhancement of DNA double-strand breaks compared to RT alone in T98G and PC-3 cells. Conclusion Our in vitro findings demonstrate that FUS has good potential to sensitize glioblastoma and prostate cancer cells to RT by mainly enhancing DNA damage.

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