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Highlights • DNA repair kinetics evaluated in T. brucei nuclear and mitochondrial genomes. • Higher efficiency of DNA repair in T. brucei cells from the mammal than the tsetse. • Differing cell cycle and survival responses to DNA damage in two T. brucei cell types. • Mitochondrial DNA repair is active in T. brucei and can involve RAD51., Genomes are affected by a wide range of damage, which has resulted in the evolution of a number of widely conserved DNA repair pathways. Most of these repair reactions have been described in the African trypanosome Trypanosoma brucei, which is a genetically tractable eukaryotic microbe and important human and animal parasite, but little work has considered how the DNA damage response operates throughout the T. brucei life cycle. Using quantitative PCR we have assessed damage induction and repair in both the nuclear and mitochondrial genomes of the parasite. We show differing kinetics of repair for three forms of DNA damage, and dramatic differences in repair between replicative life cycle forms found in the testse fly midgut and the mammal. We find that mammal-infective T. brucei cells repair oxidative and crosslink-induced DNA damage more efficiently than tsetse-infective cells and, moreover, very distinct patterns of induction and repair of DNA alkylating damage in the two life cycle forms. We also reveal robust repair of DNA lesions in the highly unusual T. brucei mitochondrial genome (the kinetoplast). By examining mutants we show that nuclear alkylation damage is repaired by the concerted action of two repair pathways, and that Rad51 acts in kinetoplast repair. Finally, we correlate repair with cell cycle arrest and cell growth, revealing that induced DNA damage has strikingly differing effects on the two life cycle stages, with distinct timing of alkylation-induced cell cycle arrest and higher levels of damage induced death in mammal-infective cells. Our data reveal that T. brucei regulates the DNA damage response during its life cycle, a capacity that may be shared by many microbial pathogens that exist in variant environments during growth and transmission.

Ovarian cancer is the most lethal gynecological malignancy. Abnormal homologous recombination repair, high level of reactive oxygen species (ROS) and upregulation of antioxidant genes are characteristic features of ovarian cancer. However, the molecular mechanisms governing the redox homeostasis in ovarian cancer cells remain to be fully elucidated. We here demonstrated a critical role of RAD51, a protein essential for homologous recombination, in the maintenance of redox homeostasis. We found that RAD51 is overexpressed in high grade serous ovarian cancer and is associated with poor prognosis. Depletion or inhibition of RAD51 results in G2/M arrest, increased production of reactive oxygen species and accumulation of oxidative DNA damage. Importantly, antioxidant N-acetylcysteine (NAC) significantly attenuated the induction of DNA damage and the perturbation of proliferation caused by RAD51 depletion. We further demonstrated that RAD51 inhibition or depletion led to elevated production of mitochondrial superoxide and increased accumulation of mitochondria. Moreover, CHK1 activation is required for the G2/M arrest and the generation of mitochondrial stress in response to RAD51 depletion. Together, our results indicate that nuclear DNA damage caused by RAD51 depletion may trigger mitochondria-originated redox dysregulation. Our findings suggest that a vicious cycle of nuclear DNA damage, mitochondrial accumulation and oxidative stress may contribute to the tumor-suppressive effects of RAD51 depletion or inhibition., Graphical abstract Image 1, Highlights • RAD51 is overexpressed in ovarian cancer and is associated with poor prognosis. • Depletion of RAD51 leads to increased mitochondrial superoxide production and oxidative DNA damage. • Increased production of mitochondrial ROS requires CHK1-mediated G2/M arrest. • mROS increase is independent of mtDNA.

Cellular oxidative stress plays a vital role in the pathological process of neural damage in cerebral ischemia/reperfusion (I/R). The breast cancer susceptibility protein 1 (BRCA1), a tumor suppressor, can modulate cellular antioxidant response and DNA repair. Yet the role of BRCA1 in cerebral I/R injury has not been explored. In this study, we observed that BRCA1 was mainly expressed in neurons and was up-regulated in response to I/R insult. Overexpression of BRCA1 attenuated reactive oxygen species production and lipid peroxidation. Enhanced BRCA1 expression promoted DNA double strand break repair through non-homologous end joining pathway. These effects consequently led to neuronal cell survival and neurological recovery. Mechanically, BRCA1 can interact with the nuclear factor (erythroid-derived 2)-like 2 (NRF2) through BRCA1 C-terminal (BRCT) domain. The cross-talk between BRCT and NRF2 activated the NRF2/Antioxidant Response Element signaling pathway and thus protected injured neurons during cerebral I/R. In conclusion, enhanced BRCA1 after cerebral I/R injury may attenuate or prevent neural damage from I/R via NRF2-mediated antioxidant pathway. The finding may provide a potential therapeutic target against ischemic stroke., Highlights • BRCA1 was up-regulated after cerebral ischemia/reperfusion injury. • Up-regulated BRCA1 attenuated cerebral ischemia/reperfusion injury and cognitive impairment. • BRCA1 binding to NRF2 via BRCT domain triggered NRF2-mediated antioxidant response. • BRCA1 promoted DSBs repair via non-homologous end joining-pathway.

Rare pleiotropic genetic disorders, Ataxia-telangiectasia (A-T), Bloom syndrome (BS) and Nijmegen breakage syndrome (NBS) are characterised by immunodeficiency, extreme radiosensitivity, higher cancer susceptibility, premature aging, neurodegeneration and insulin resistance. Some of these functional abnormalities can be explained by aberrant DNA damage response and chromosomal instability. It has been suggested that one possible common denominator of these conditions could be chronic oxidative stress caused by endogenous ROS overproduction and impairment of mitochondrial homeostasis. Recent studies indicate new, alternative sources of oxidative stress in A-T, BS and NBS cells, including NADPH oxidase 4 (NOX4), oxidised low-density lipoprotein (ox-LDL) or Poly (ADP-ribose) polymerases (PARP). Mitochondrial abnormalities such as changes in the ultrastructure and function of mitochondria, excess mROS production as well as mitochondrial damage have also been reported in A-T, BS and NBS cells. A-T, BS and NBS cells are inextricably linked to high levels of reactive oxygen species (ROS), and thereby, chronic oxidative stress may be a major phenotypic hallmark in these diseases. Due to the presence of mitochondrial disturbances, A-T, BS and NBS may be considered mitochondrial diseases. Excess activity of antioxidant enzymes and an insufficient amount of low molecular weight antioxidants indicate new pharmacological strategies for patients suffering from the aforementioned diseases. However, at the current stage of research we are unable to ascertain if antioxidants and free radical scavengers can improve the condition or prolong the survival time of A-T, BS and NBS patients. Therefore, it is necessary to conduct experimental studies in a human model., Graphical abstract fx1

Replication perturbations activate DNA damage tolerance (DDT) pathways, which are crucial to promote replication completion and to prevent fork breakage, a leading cause of genome instability. One mode of DDT uses translesion synthesis polymerases, which however can also introduce mutations. The other DDT mode involves recombination-mediated mechanisms, which are generally accurate. DDT occurs prevalently postreplicatively, but in certain situations homologous recombination is needed to restart forks. Fork reversal can function to stabilize stalled forks, but may also promote error-prone outcome when used for fork restart. Recent years have witnessed important advances in our understanding of the mechanisms and DNA structures that mediate recombination-mediated damage-bypass and highlighted principles that regulate DDT pathway choice locally and temporally. In this review we summarize the current knowledge and paradoxes on recombination-mediated DDT pathways and their workings, discuss how the intermediate DNA structures may influence genome integrity, and outline key open questions for future research.

Glucocorticoids (GCs), which act on stress pathways, are well-established in the co-treatment of different kinds of tumors; however, the underlying mechanisms by which GCs act are not yet well elucidated. As such, this work investigates the role of glucocorticoids, specifically dexamethasone (DEXA), in the processes referred to as DNA damage and DNA damage response (DDR), establishing a new approach in three astrocytomas cell lines (CT2A, APP.PS1 L.1 and APP.PS1 L.3). The results show that DEXA administration increased the basal levels of gamma-H2AX foci, keeping them higher 4 h after irradiation (IR) of the cells, compared to untreated cells. This means that DEXA might cause increased radiosensitivity in these cell lines. On the other hand, DEXA did not have an apparent effect on the formation and disappearance of the 53BP1 foci. Furthermore, it was found that DEXA administered 2 h before IR led to a radical change in DNA repair kinetics, even DEXA does not affect cell cycle. It is important to highlight that DEXA produced cell death in these cell lines compared to untreated cells. Finally and most important, the high levels of gamma-H2AX could be reversed by administration of ascorbic acid, a potent blocker of reactive oxygen species, suggesting that DEXA acts by causing DNA damage via oxidative stress. These exiting findings suggest that DEXA might promote radiosensitivity in brain tumors, specifically in astrocytoma-like tumors., Graphical abstract, Highlights ● Dexamethasone causes DNA damage by increasing gamma-H2AX levels in three astrocytoma cell lines (CT2A, APP.PS1 L.1 and APP.PS1 L.3) ● Dexamethasone affects DNA repair kinetics and produces cell death in three astrocytoma cell lines (CT2A, APP.PS1 L.1 and APP.PS1 L.3) even dexamethasone does not affect any cell cycle arrest in any cell line studied. ● Oxidative stress appears to be one of the mechanisms of dexamethasone action in DNA damage as their effect is reversed with ascorbic acid addition.

Huntington disease (HD) is a neurodegenerative disease caused by expansion of CAG repeats in the huntingtin (Htt) gene. The expression of hMTH1, the human hydrolase that degrades oxidized purine nucleoside triphosphates, grants protection in a chemical HD mouse model in which HD-like features are induced by the mitochondrial toxin 3-nitropropionic acid (3-NP). To further examine the relationship between oxidized dNTPs and HD-like neurodegeneration, we studied the effects of hMTH1 expression in a genetic cellular model for HD, such as striatal cells expressing mutant htt (HdhQ111). hMTH1 expression protected these cells from 3-NP and H2O2-induced killing, by counteracting the mutant htt-dependent increased vulnerability and accumulation of nuclear and mitochondrial DNA 8-hydroxyguanine levels. hMTH1 expression reverted the decreased mitochondrial membrane potential characteristic of HdhQ111 cells and delayed the increase in mitochondrial reactive oxygen species associated with 3-NP treatment. Further indications of hMTH1-mediated mitochondrial protection are the partial reversion of 3-NP-induced alterations in mitochondrial morphology and the modulation of DRP1 and MFN1 proteins, which control fusion/fission rates of mitochondria. Finally, in line with the in vitro findings, upon 3-NP in vivo treatment, 8-hydroxyguanine levels in mitochondrial DNA from heart, muscle and brain are significantly lower in transgenic hMTH1-expressing mice than in wild-type animals., Highlights ► hMTH1 provides defence against cell death in mutant htt-expressing striatal cells ► hMTH1 improves mitochondrial functionality in mutant htt-expressing striatal cells ► hMTH1 protects in vivo mitochondria from endogenous and 3-NP induced oxidation