DNA double-strand breaks (DSBs), such as those produced by radiation and radiomimetics, are amongst the most toxic forms of cellular damage, in part because they involve extensive oxidative modifications at the break termini. Prior to completion of DSB repair, the chemically modified termini must be removed. Various DNA processing enzymes have been implicated in the processing of these dirty ends, but molecular knowledge of this process is limited. Here, we demonstrate a role for the metallo-β-lactamase fold 5'-3' exonuclease SNM1A in this vital process. Cells disrupted for SNM1A manifest increased sensitivity to radiation and radiomimetic agents and show defects in DSB damage repair. SNM1A is recruited and is retained at the sites of DSB damage via the concerted action of its three highly conserved PBZ, PIP box and UBZ interaction domains, which mediate interactions with poly-ADP-ribose chains, PCNA and the ubiquitinated form of PCNA, respectively. SNM1A can resect DNA containing oxidative lesions induced by radiation damage at break termini. The combined results reveal a crucial role for SNM1A to digest chemically modified DNA during the repair of DSBs and imply that the catalytic domain of SNM1A is an attractive target for potentiation of radiotherapy.

• MeSH
• DNA metabolismus   genetika   MeSH
• dvouřetězcové zlomy DNA * účinky záření   MeSH
• enzymy opravy DNA * metabolismus   genetika   MeSH
• exodeoxyribonukleasy * metabolismus   genetika   MeSH
• lidé MeSH
• oprava DNA * MeSH
• proliferační antigen buněčného jádra metabolismus   genetika   MeSH
• proteiny buněčného cyklu MeSH
• ubikvitinace MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DCLRE1A protein, human MeSH Prohlížeč
• DNA MeSH
• enzymy opravy DNA * MeSH
• exodeoxyribonukleasy * MeSH
• PCNA protein, human MeSH Prohlížeč
• proliferační antigen buněčného jádra MeSH
• proteiny buněčného cyklu MeSH

BACKGROUND: There is currently significant interest in assessing the role of oxygen in the radiobiological effects at ultra-high dose rates. Oxygen modulation is postulated to play a role in the enhanced sparing effect observed in FLASH radiotherapy, where particles are delivered at 40-1000 Gy/s. Furthermore, the development of laser-driven accelerators now enables radiobiology experiments in extreme regimes where dose rates can exceed 109 Gy/s, and predicted oxygen depletion effects on cellular response can be tested. Access to appropriate experimental enviroments, allowing measurements under controlled oxygenation conditions, is a key requirement for these studies. We report on the development and application of a bespoke portable hypoxia chamber specifically designed for experiments employing laser-driven sources, but also suitable for comparator studies under FLASH and conventional irradiation conditions. MATERIALS AND METHODS: We used oxygen concentration measurements to test the induction of hypoxia and the maintenance capacity of the chambers. Cellular hypoxia induction was verified using hypoxia inducible factor-1α immunostaining. Calibrated radiochromic films and GEANT-4 simulations verified the dosimetry variations inside and outside the chambers. We irradiated hypoxic human skin fibroblasts (AG01522B) cells with laser-driven protons, conventional protons and reference 225 kVp X-rays to quantify DNA DSB damage and repair under hypoxia. We further measured the oxygen enhancement ratio for cell survival after X-ray exposure in normal fibroblast and radioresistant patient- derived GBM stem cells. RESULTS: Oxygen measurements showed that our chambers maintained a radiobiological hypoxic environment for at least 45 min and pathological hypoxia for up to 24 h after disconnecting the chambers from the gas supply. We observed a significant reduction in the 53BP1 foci induced by laser-driven protons, conventional protons and X-rays in the hypoxic cells compared to normoxic cells at 30 min post-irradiation. Under hypoxic irradiations, the Laser-driven protons induced significant residual DNA DSB damage in hypoxic AG01522B cells compared to the conventional dose rate protons suggesting an important impact of these extremely high dose-rate exposures. We obtained an oxygen enhancement ratio (OER) of 2.1 ± 0.1 and 2.5 ± 0.1 respectively for the AG01522B and patient-derived GBM stem cells for X-ray irradiation using our hypoxia chambers. CONCLUSION: We demonstrated the design and application of portable hypoxia chambers for studying cellular radiobiological endpoints after exposure to laser-driven protons at ultra-high dose, conventional protons and X-rays. Suitable levels of reduced oxygen concentration could be maintained in the absence of external gassing to quantify hypoxic effects. The data obtained provided indication of an enhanced residual DNA DSB damage under hypoxic conditions at ultra-high dose rate compared to the conventional protons or X-rays.

• Klíčová slova
• DNA repair, Hypoxia, Laser-driven protons, Ultra-high dose rate,
• MeSH
• DNA účinky záření   MeSH
• hypoxie MeSH
• kyslík MeSH
• lasery MeSH
• lidé MeSH
• protony * MeSH
• radiobiologie * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA MeSH
• kyslík MeSH
• protony * MeSH

DNA double-strand breaks (DSBs) have been recognized as the most serious lesions in irradiated cells. While several biochemical pathways capable of repairing these lesions have been identified, the mechanisms by which cells select a specific pathway for activation at a given DSB site remain poorly understood. Our knowledge of DSB induction and repair has increased dramatically since the discovery of ionizing radiation-induced foci (IRIFs), initiating the possibility of spatiotemporally monitoring the assembly and disassembly of repair complexes in single cells. IRIF exploration revealed that all post-irradiation processes-DSB formation, repair and misrepair-are strongly dependent on the characteristics of DSB damage and the microarchitecture of the whole affected chromatin domain in addition to the cell status. The microscale features of IRIFs, such as their morphology, mobility, spatiotemporal distribution, and persistence kinetics, have been linked to repair mechanisms. However, the influence of various biochemical and structural factors and their specific combinations on IRIF architecture remains unknown, as does the hierarchy of these factors in the decision-making process for a particular repair mechanism at each individual DSB site. New insights into the relationship between the physical properties of the incident radiation, chromatin architecture, IRIF architecture, and DSB repair mechanisms and repair efficiency are expected from recent developments in optical superresolution microscopy (nanoscopy) techniques that have shifted our ability to analyze chromatin and IRIF architectures towards the nanoscale. In the present review, we discuss this relationship, attempt to correlate still rather isolated nanoscale studies with already better-understood aspects of DSB repair at the microscale, and consider whether newly emerging "correlated multiscale structuromics" can revolutionarily enhance our knowledge in this field.

• Klíčová slova
• DNA damage and repair, DNA double-strand breaks (DSBs), DSB repair pathway choice and hierarchy, chromatin architecture, ionizing radiation, ionizing radiation-induced foci (IRIFs), linear energy transfer (LET), single-molecule localization microscopy (SMLM), superresolution microscopy,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Nuclear architecture plays a significant role in DNA repair mechanisms. It is evident that proteins involved in DNA repair are compartmentalized in not only spontaneously occurring DNA lesions or ionizing radiation-induced foci (IRIF), but a specific clustering of these proteins can also be observed within the whole cell nucleus. For example, 53BP1-positive and BRCA1-positive DNA repair foci decorate chromocenters and can appear close to nuclear speckles. Both 53BP1 and BRCA1 are well-described factors that play an essential role in double-strand break (DSB) repair. These proteins are members of two protein complexes: 53BP1-RIF1-PTIP and BRCA1-CtIP, which make a "decision" determining whether canonical nonhomologous end joining (NHEJ) or homology-directed repair (HDR) is activated. It is generally accepted that 53BP1 mediates the NHEJ mechanism, while HDR is activated via a BRCA1-dependent signaling pathway. Interestingly, the 53BP1 protein appears relatively quickly at DSB sites, while BRCA1 is functional at later stages of DNA repair, as soon as the Mre11-Rad50-Nbs1 complex is recruited to the DNA lesions. A function of the 53BP1 protein is also linked to a specific histone signature, including phosphorylation of histone H2AX (γH2AX) or methylation of histone H4 at the lysine 20 position (H4K20me); therefore, we also discuss an epigenetic landscape of 53BP1-positive DNA lesions.

• Klíčová slova
• 53BP1, BRCA1, DNA damage, epigenetics, histone modifications,
• MeSH
• 53BP1 genetika   metabolismus   MeSH
• buněčné jádro genetika   metabolismus   MeSH
• fosforylace MeSH
• lidé MeSH
• oprava DNA * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• 53BP1 MeSH

Repair of ribosomal DNA (rDNA) is a very important nuclear process due to the most active transcription of ribosomal genes. Proper repair of rDNA is required for physiological biogenesis of ribosomes. Here, we analyzed the epigenetics of the DNA damage response in a nucleolar compartment, thus in the ribosomal genes studied in nonirradiated and UVA-irradiated mouse embryonic fibroblasts (MEFs). We found that the promoter of ribosomal genes is not abundant on H4K20me2, but it is densely occupied by H4K20me3. Ribosomal genes, regulated via UBF1/2 proteins, were characterized by an interaction between UBF1/2 and H4K20me2/me3. This interaction was strengthened by UVA irradiation that additionally causes a focal accumulation of H4K20me3 in the nucleolus. No interaction has been found between UBF1/2 and H3K9me3. Interestingly, UVA irradiation decreases the levels of H3K9me3 and H4K20me3 at 28S rDNA. Altogether, the UVA light affects the epigenetic status of ribosomal genes at 28S rDNA and strengthens an interaction between UBF1/2 proteins and H4K20me2/me3.

• Klíčová slova
• DNA damage response, DNA repair, Nucleolus, UBF, UVA irradiation,
• MeSH
• buněčné jadérko metabolismus   MeSH
• buněčné jádro metabolismus   MeSH
• chromatinová imunoprecipitace MeSH
• DNA vazebné proteiny MeSH
• epigeneze genetická účinky záření   MeSH
• fluorescenční protilátková technika MeSH
• histony metabolismus   MeSH
• metylace MeSH
• myši MeSH
• promotorové oblasti (genetika) MeSH
• regulace genové exprese účinky záření   MeSH
• ribozomální DNA genetika   MeSH
• transkripční iniciační komplex Pol1 - proteiny metabolismus   MeSH
• ultrafialové záření * MeSH
• vazba proteinů MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• histony MeSH
• ribozomální DNA MeSH
• transcription factor UBF MeSH Prohlížeč
• transkripční iniciační komplex Pol1 - proteiny MeSH

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.

• Klíčová slova
• 53BP1, 53BP1, p53 binding protein 1, ATM, ataxia telangiectasia mutated kinase, BRCA1, breast cancer type 1 susceptibility protein, Cdk, cyclin dependent kinase, DDR, DNA damage response, DNA damage response, H2AX, histone variant H2AX, IR – ionizing radiation, MDC1, mediator of DNA damage checkpoint protein 1, NCS – neocarzinostatin, NZ – nocodazole, PTIP, PAX transactivation activation domain-interacting protein, Plk1, Polo-like kinase 1, Polo like kinase 1, RIF1, Rap1-interacting factor 1 homolog, RNAi, RNA interference, RNF168, RING finger protein 168, RNF8, RING finger protein 8, mitosis, phosphorylation,
• MeSH
• 53BP1 MeSH
• fosforylace MeSH
• HeLa buňky MeSH
• histony metabolismus   MeSH
• intracelulární signální peptidy a proteiny antagonisté a inhibitory   chemie   metabolismus   MeSH
• kinetochory metabolismus   MeSH
• lidé MeSH
• malá interferující RNA metabolismus   MeSH
• mitóza * MeSH
• nádorové buněčné linie MeSH
• oprava DNA * MeSH
• polo-like kinasa 1 MeSH
• poškození DNA účinky záření   MeSH
• protein-serin-threoninkinasy antagonisté a inhibitory   genetika   metabolismus   MeSH
• proteinkinasa CDC2 metabolismus   MeSH
• proteiny buněčného cyklu antagonisté a inhibitory   genetika   metabolismus   MeSH
• protoonkogenní proteiny antagonisté a inhibitory   genetika   metabolismus   MeSH
• RNA interference MeSH
• terciární struktura proteinů MeSH
• ubikvitinace MeSH
• záření gama MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 53BP1 MeSH
• histony MeSH
• intracelulární signální peptidy a proteiny MeSH
• malá interferující RNA MeSH
• protein-serin-threoninkinasy MeSH
• proteinkinasa CDC2 MeSH
• proteiny buněčného cyklu MeSH
• protoonkogenní proteiny MeSH
• TP53BP1 protein, human MeSH Prohlížeč

Error-free repair of DNA double-strand breaks (DSBs) is achieved by homologous recombination (HR), and BRCA1 is an important factor for this repair pathway. In the absence of BRCA1-mediated HR, the administration of PARP inhibitors induces synthetic lethality of tumour cells of patients with breast or ovarian cancers. Despite the benefit of this tailored therapy, drug resistance can occur by HR restoration. Genetic reversion of BRCA1-inactivating mutations can be the underlying mechanism of drug resistance, but this does not explain resistance in all cases. In particular, little is known about BRCA1-independent restoration of HR. Here we show that loss of REV7 (also known as MAD2L2) in mouse and human cell lines re-establishes CTIP-dependent end resection of DSBs in BRCA1-deficient cells, leading to HR restoration and PARP inhibitor resistance, which is reversed by ATM kinase inhibition. REV7 is recruited to DSBs in a manner dependent on the H2AX-MDC1-RNF8-RNF168-53BP1 chromatin pathway, and seems to block HR and promote end joining in addition to its regulatory role in DNA damage tolerance. Finally, we establish that REV7 blocks DSB resection to promote non-homologous end-joining during immunoglobulin class switch recombination. Our results reveal an unexpected crucial function of REV7 downstream of 53BP1 in coordinating pathological DSB repair pathway choices in BRCA1-deficient cells.

• MeSH
• 53BP1 MeSH
• adaptorové proteiny signální transdukční MeSH
• ATM protein antagonisté a inhibitory   metabolismus   MeSH
• buněčné linie MeSH
• chemorezistence genetika   MeSH
• chromatin metabolismus   MeSH
• chromozomální proteiny, nehistonové metabolismus   MeSH
• DNA vazebné proteiny metabolismus   MeSH
• dvouřetězcové zlomy DNA * MeSH
• histony metabolismus   MeSH
• intracelulární signální peptidy a proteiny metabolismus   MeSH
• jaderné proteiny metabolismus   MeSH
• lidé MeSH
• Mad2 protein nedostatek   genetika   metabolismus   MeSH
• myši MeSH
• PARP inhibitory * MeSH
• přesmyk imunoglobulinových tříd genetika   MeSH
• protein BRCA1 nedostatek   genetika   metabolismus   MeSH
• proteiny buněčného cyklu MeSH
• rekombinační oprava DNA * MeSH
• trans-aktivátory metabolismus   MeSH
• ubikvitinligasy metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 53BP1 MeSH
• adaptorové proteiny signální transdukční MeSH
• ATM protein MeSH
• chromatin MeSH
• chromozomální proteiny, nehistonové MeSH
• DNA vazebné proteiny MeSH
• H2AX protein, human MeSH Prohlížeč
• histony MeSH
• intracelulární signální peptidy a proteiny MeSH
• jaderné proteiny MeSH
• Mad2 protein MeSH
• MAD2L2 protein, human MeSH Prohlížeč
• Mad2l2 protein, mouse MeSH Prohlížeč
• MDC1 protein, human MeSH Prohlížeč
• PARP inhibitory * MeSH
• protein BRCA1 MeSH
• proteiny buněčného cyklu MeSH
• RNF168 protein, human MeSH Prohlížeč
• RNF8 protein, human MeSH Prohlížeč
• TP53BP1 protein, human MeSH Prohlížeč
• trans-aktivátory MeSH
• Trp53bp1 protein, mouse MeSH Prohlížeč
• ubikvitinligasy MeSH