The role of ATM signalling and its mediator proteins in DNA double strand break repair

Although most DNA double strand breaks (DSBs) are repaired by DNA nonhomologous end!joining (NHEJ), DSBs at heterochromatin (HC) regions undergo repair by homologous recombination (HR) in G2 phase. Repair of DSBs at HC regions requires ATM-dependent KAP1 phosphorylation and subsequent HC relaxation. The mediator proteins facilitate DSB repair at HC in G1 phase by retaining ATM and hence pKAP1 at DSBs until the completion of repair. In this thesis, I investigated the role of the mediator proteins in enabling DSB repair in G2 phase. I demonstrate that the mediator proteins are required for the slow component of DSB repair in G2, which represents HR. They also promote ATM-dependent pKAP1 formation in G2 as in G1. In addition, I have described a role for MDC1 in Rad51 loading and for RNF8 in DNA resection. Moreover, I demonstrate that BRCA1 overcomes an inhibitory barrier by 53BP1 to resection by promoting a G2!specific enlargement in 53BP1 foci during HR that involves 53BP1 repositioning to the foci periphery and vacation from the central core. RPA foci form in the core devoid of 53BP1. 53BP1 has opposing roles in HR; it creates a restrictive barrier to resection but promotes pKAP1 and HC relaxation. RAP80 also inhibits resection by binding to ubiquitylated histones at DSBs. I demonstrate that the DUB enzyme, POH1, is required to overcome the barrier posed to resection by RAP80 since its depletion leads to deficient 53BP1 vacation of the central core and deficient resection. BRCA1 and POH1 cooperate during G2 phase to promote resection and DSB repair by HR. Additionally; I investigated the role(s) of the chromatin remodelers BAF180 and CHD7 in transcriptional silencing following DSB induction, a process that requires ATM, RNF8 and RNF168. I demonstrate that deficient transcriptional silencing leads to a DSB repair defect at early times post IR

Impacts of prolonged exposure to low concentration of titanium dioxide nanoparticles on cell cycle control and DNA repair

Although the toxicological profile of titanium dioxide nanoparticles is not fully illuminated, large quantities of titanium dioxide nanoparticles (TiO2NPs) are now produced. In our study, we evaluated the cytotoxic and genotoxic impacts of titanium dioxide nanoparticles on different cell lines (normal, cancer and DNA repair-deficient cells). MTT assay was used to evaluate the cytotoxicity, γ-H2AX and 53BP1 assay was used to evaluate the genotoxicity and G2/M assay was used to study the impacts of titanium dioxide nanoparticles on cell cycle regulation. In this study normal and DNA repair-deficient cell lines were used to study the repair mechanism of titanium dioxide nanoparticles induced DNA damage. G2/M checkpoint maintenance was also evaluated. We demonstrate that prolonged exposure to low concentrations of titanium dioxide nanoparticles does not induce significant cytotoxicity but induces significant genotoxicity, particularly DNA double-strand breaks (DNA DSBs). Furthermore, this study demonstrated that DNA DSBs at heterochromatin region are ATM-dependent and DNA DSBs at euchromatin region are ATM-independent and DNA PKcs dependent. After exposure to titanium dioxide nanoparticles, we show that the activation of G2/M checkpoint is DNA DSBs dependent threshold as does checkpoint release. All in all, we showed that prolonged exposure to low concentrations of titanium dioxide nanoparticles does not affect cell viability but causes DNA damage and cell cycle checkpoint adaptation which may lead to genetic instability. DOI: http://dx.doi.org/10.5281/zenodo.748742

DNA DSB repair pathway choice: an orchestrated handover mechanism

DNA double strand breaks (DSBs) are potential lethal lesions but can also lead to chromosome rearrangements, a step promoting carcinogenesis. DNA non-homologous end-joining (NHEJ) is the major DSB rejoining process and occurs in all cell cycle stages. Homologous recombination (HR) can additionally function to repair irradiation-induced two-ended DSBs in G2 phase. In mammalian cells, HR predominantly uses a sister chromatid as a template for DSB repair; thus HR functions only in late S/G2 phase. Here, we review current insight into the interplay between HR and NHEJ in G2 phase. We argue that NHEJ represents the first choice pathway, repairing approximately 80% of X-ray-induced DSBs with rapid kinetics. However, a subset of DSBs undergoes end resection and repair by HR. 53BP1 restricts resection, thereby promoting NHEJ. During the switch fromNHEJ to HR, 53BP1 is repositioned to the periphery of enlarged irradiation-induced foci (IRIF) via a BRCA1-dependent process. K63-linked ubiquitin chains, which also form at IRIF, are also repositioned as well as receptor-associated protein 80 (RAP80), a ubiquitin binding protein. RAP80 repositioning requires POH1, a proteasome component. Thus, the interfacing barriers to HR, 53BP1 and RAP80 are relieved by POH1 and BRCA1, respectively. Removal of RAP80 from the IRIF core is required for loss of the ubiquitin chains and 53BP1, and for efficient replication protein A foci formation. We propose that NHEJ is used preferentially to HR because it is a compact process that does not necessitate extensive chromatin changes in the DSB vicinity

HOTEL FRONT LINE EMPLOYEES’ PERCEPTIONS ON LEADERSHIP AND WORKPLACE MOTIVATION IN TIMES OF CRISIS

Purpose - This study aims to identify the extent to which different motivational elements can support strong bonds and good cooperation between front-line employees and hotels’ leadership in times of crisis and work suspension. Design - The views of a sample of employees working in 4 and 5 star hotels in Heraklion Prefecture were studied in May 2020, when hotel operations were suspended. Methodology - Two hundred and one completed questionnaires were collected, consisting of 31 six-point Likert scales, whose data were analysed using the Exploratory and Confirmatory Factor Analysis (CFA). Approach - The Exploratory Factor Analysis (EFA) followed the principal component method of extraction based on a varimax rotation, while the CFA adοpted the method of Structural Equation Modeling, that was based on the maximum likelihood method of estimation. Results - In times of crisis, front-line employees of hotels consider that the creation of strong bonds and a good spirit of cooperation and communication between them and the leadership of the hotels is strongly based on the exploitation of their skills and the developmental opportunities offered. Originality of the research - The research identified motivational factors that can support the creation of strong bonds and a good spirit of cooperation and communication between front-line employees and hotel management in times of crisis. On a practical level, the findings can help hotel managers gain strategic advantage and experience for the future, should they need to deal with similar situations

SETDB1, HP1 and SUV39 promote repositioning of 53BP1 to extend resection during homologous recombination in G2 cells

Recent studies have shown that homologous recombination (HR) requires chromatin repression as well as relaxation at DNA double strand breaks (DSBs). HP1 and SUV39H1/2 are repressive factors essential for HR. Here, we identify SETDB1 as an additional compacting factor promoting HR. Depletion of HP1, SUV39, SETDB1 or BRCA1 confer identical phenotypes. The repressive factors, like BRCA1, are dispensable for the initiation of resection but promote the extension step causing diminished RPA or RAD51 foci and HR in irradiated G2 cells. Depletion of the compacting factors does not inhibit BRCA1 recruitment but at 8 h post IR, BRCA1 foci are smaller and aberrantly positioned compared to control cells. BRCA1 promotes 53BP1 repositioning to the periphery of enlarged foci and formation of a devoid core with BRCA1 becoming enlarged and localised internally to 53BP1. Depletion of the compacting factors precludes these changes at irradiation-induced foci. Thus, the repressive factors are required for BRCA1 function in promoting the repositioning of 53BP1 during HR. Additionally, depletion of these repressive factors in undamaged cells causes diminished sister chromatid association at centromeric sequences. We propose a model for how these findings may be functionally linked

Co-operation of BRCA1 and POH1 relieves the barriers posed by 53BP1 and RAP80 to resection

In G2 phase cells, DNA double-strand break repair switches from DNA non-homologous end-joining to homologous recombination. This switch demands the promotion of resection. We examine the changes in 53BP1 and RAP80 ionizing radiation induced foci (IRIF) in G2 phase, as these are factors that restrict resection. We observed a 2-fold increase in the volume of 53BP1 foci by 8 h, which is not seen in G1 cells. Additionally, an IRIF core devoid of 53BP1 arises where RPA foci form, with BRCA1 IRIF forming between 53BP1 and replication protein A (RPA). Ubiquitin chains assessed using a-FK2 antibodies are similarly repositioned. Repositioning of all these components requires BRCA1’s BRCT but not the ring finger domain. 53BP1, RAP80 and ubiquitin chains are enlarged following POH1 depletion by small interfering RNA, but a devoid core does not form and RPA foci formation is impaired. Co-depletion of POH1 and RAP80, BRCC36 or ABRAXAS allows establishment of the 53BP1 and ubiquitin chain-devoid core. Thus, the barriers posed by 53BP1 and RAP80 are relieved by BRCA1 and POH1, respectively. Analysis of combined depletions shows that these represent distinct but interfacing barriers to promote loss of ubiquitin chains in the IRIF core, which is required for subsequent resection. We propose a model whereby BRCA1 impacts on 53BP1 to allow access of POH1 to RAP80. POH1-dependent removal of RAP80 within the IRIF core enables degradation of ubiquitin chains, which promotes loss of 53BP1. Thus, POH1 represents a novel component regulating the switch from nonhomologous end-joining to homologous recombination

Requirement for PBAF in transcriptional repression and repair at DNA breaks in actively transcribed regions of chromatin

Actively transcribed regions of the genome are vulnerable to genomic instability. Recently, it was discovered that transcription is repressed in response to neighboring DNA double-strand breaks (DSBs). It is not known whether a failure to silence transcription flanking DSBs has any impact on DNA repair efficiency or whether chromatin remodelers contribute to the process. Here, we show that the PBAF remodeling complex is important for DSB-induced transcriptional silencing and promotes repair of a subset of DNA DSBs at early time points, which can be rescued by inhibiting transcription globally. An ATM phosphorylation site on BAF180, a PBAF subunit, is required for both processes. Furthermore, we find that subunits of the PRC1 and PRC2 polycomb group complexes are similarly required for DSB-induced silencing and promoting repair. Cancer-associated BAF180 mutants are unable to restore these functions, suggesting PBAF's role in repressing transcription near DSBs may contribute to its tumor suppressor activity

How cancer cells hijack DNA double-strand break repair pathways to gain genomic instability

DNA double-strand breaks (DSBs) are a significant threat to the viability of a normal cell, since they can result in loss of genetic material if mitosis or replication is attempted in their presence. Consequently, evolutionary pressure has resulted in multiple pathways and responses to enable DSBs to be repaired efficiently and faithfully. Cancer cells, which are under pressure to gain genomic instability, have a striking ability to avoid the elegant mechanisms by which normal cells maintain genomic stability. Current models suggest that in normal cells DSB repair occurs in a hierarchical manner that promotes rapid and efficient rejoining first, with the utilisation of additional steps or pathways of diminished accuracy if rejoining is unsuccessful or delayed. We evaluate the fidelity of DSB repair pathways and discuss how cancer cells promote the utilisation of less accurate processes. Homologous recombination serves to promote accuracy and stability during replication, providing a battlefield for cancer to gain instability. Non-homologous end-joining, a major DSB repair pathway in mammalian cells, usually operates with high fidelity and only switches to less faithful modes if timely repair fails. The transition step is finely tuned and provides another point of attack during tumour progression. In addition to DSB repair, a DSB signalling response activates processes such as cell cycle checkpoint arrest, which enhance the possibility of accurate DSB repair. We will consider the ways by which cancers modify and accost these processes to gain genomic instabilit

Chromatin organization revealed by nanostructure of irradiation induced gamma H2AX, 53BP1 and Rad51 foci

The spatial distribution of DSB repair factors gamma H2AX, 53BP1 and Rad51 in ionizing radiation induced foci (IRIF) in HeLa cells using super resolution STED nanoscopy after low and high linear energy transfer (LET) irradiation was investigated. 53BP1 and gamma H2AX form IRIF with same mean size of (540 +/- 40) nm after high LET irradiation while the size after low LET irradiation is significantly smaller. The IRIF of both repair factors show nanostructures with partial anti-correlation. These structures are related to domains formed within the chromatin territories marked by gamma H2AX while 53BP1 is mainly situated in the perichromatin region. The nanostructures have a mean size of (129 +/- 6) nm and are found to be irrespective of the applied LET and the labelled damage marker. In contrast, Rad51 shows no nanostructure and a mean size of (143 +/- 13) nm independent of LET. Although Rad51 is surrounded by 53BP1 it strongly anti-correlates meaning an exclusion of 53BP1 next to DSB when decision for homologous DSB repair happened

Opposing roles for 53BP1 during homologous recombination

Although DNA non-homologous end-joining repairs most DNA double-strand breaks (DSBs) in G2 phase, late repairing DSBs undergo resection and repair by homologous recombination (HR). Based on parallels to the situation in G1 cells, previous work has suggested that DSBs that undergo repair by HR predominantly localize to regions of heterochromatin (HC). By using H3K9me3 and H4K20me3 to identify HC regions, we substantiate and extend previous evidence, suggesting that HC-DSBs undergo repair by HR. Next, we examine roles for 53BP1 and BRCA1 in this process. Previous studies have shown that 53BP1 is pro-non-homologous end-joining and anti-HR. Surprisingly, we demonstrate that in G2 phase, 53BP1 is required for HR at HC-DSBs with its role being to promote phosphorylated KAP-1 foci formation. BRCA1, in contrast, is dispensable for pKAP-1 foci formation but relieves the barrier caused by 53BP1. As 53BP1 is retained at irradiation-induced foci during HR, we propose that BRCA1 promotes displacement but retention of 53BP1 to allow resection and any necessary HC modifications to complete HR. In contrast to this role for 53BP1 in HR in G2 phase, we show that it is dispensable for HR in S phase, where HC regions are likely relaxed during replication