DNA polymerase eta protects human cells against DNA damage induced by the tumor chemotherapeutic temozolomide.

Human DNA polymerases can bypass DNA lesions performing translesion synthesis (TLS), a mechanism of DNA damage tolerance. Tumor cells use this mechanism to survive lesions caused by specific chemotherapeutic agents, resulting in treatment relapse. Moreover, TLS polymerases are error-prone and, thus, can lead to mutagenesis, increasing the resistance potential of tumor cells. DNA polymerase eta (pol eta) - a key protein from this group - is responsible for protecting against sunlight-induced tumors. Xeroderma Pigmentosum Variant (XP-V) patients are deficient in pol eta activity, which leads to symptoms related to higher sensitivity and increased incidence of skin cancer. Temozolomide (TMZ) is a chemotherapeutic agent used in glioblastoma and melanoma treatment. TMZ damages cells' genomes, but little is known about the role of TLS in TMZ-induced DNA lesions. This work investigates the effects of TMZ treatment in human XP-V cells, which lack pol eta, and in its complemented counterpart (XP-V comp). Interestingly, TMZ reduces the viability of XP-V cells compared to TLS proficient control cells. Furthermore, XP-V cells treated with TMZ presented increased phosphorylation of H2AX, forming γH2AX, compared to control cells. However, cell cycle assays indicate that XP-V cells treated with TMZ replicate damaged DNA and pass-through S-phase, arresting in the G2/M-phase. DNA fiber assay also fails to show any specific effect of TMZ-induced DNA damage blocking DNA elongation in pol eta deficient cells. These results show that pol eta plays a role in protecting human cells from TMZ-induced DNA damage, but this can be different from its canonical TLS mechanism. The new role opens novel therapeutic possibilities of using pol eta as a target to improve the efficacy of TMZ-based therapies against cancer.

RHOAming Through the Nucleotide Excision Repair Pathway as a Mechanism of Cellular Response Against the Effects of UV Radiation.

Typical Rho GTPases include the enzymes RhoA, Rac1, and Cdc42 that act as molecular switches to regulate essential cellular processes in eukaryotic cells such as actomyosin dynamics, cell cycle, adhesion, death and differentiation. Recently, it has been shown that different conditions modulate the activity of these enzymes, but their functions still need to be better understood. Here we examine the interplay between RhoA and the NER (Nucleotide Excision Repair) pathway in human cells exposed to UVA, UVB or UVC radiation. The results show high levels and accumulation of UV-induced DNA lesions (strand breaks and cyclobutane pyrimidine dimers, CPDs) in different cells with RhoA loss of function (LoF), either by stable overexpression of negative dominant RhoA (RhoA-N19 mutant), by inhibition with C3 toxin or by transient silencing with siRNA. Cells under RhoA LoF showed reduced levels of γH2AX, p-Chk1 (Ser345) and p-p53 (Ser15) that reflected causally in their accumulation in G1/S phases, in low survival rates and in reduced cell proliferation, also in accordance with the energy of applied UV light. Even NER-deficient cells (XPA, XPC) or DNA translesion synthesis (TLS)-deficient cells (XPV) showed substantial hypersensitivity to UV effects when previously submitted to RhoA LoF. In contrast, analyses of apoptosis, necrosis, autophagy and senescence revealed that all cells displaying normal levels of active RhoA (RhoA-GTP) are more resistant to UV-promoted cell death. This work reaffirms the role of RhoA protein signaling in protecting cells from damage caused by UV radiation and demonstrates relevant communicating mechanisms between actin cytoskeleton and genomic stability.

XPD/ERCC2 mutations interfere in cellular responses to oxidative stress.

Nucleotide excision repair (NER) is a conserved, flexible mechanism responsible for the removal of bulky, helix-distorting DNA lesions, like ultraviolet damage or cisplatin adducts, but its role in the repair of lesions generated by oxidative stress is still not clear. The helicase XPD/ERCC2, one of the two helicases of the transcription complex IIH, together with XPB, participates both in NER and in RNA pol II-driven transcription. In this work, we investigated the responses of distinct XPD-mutated cell lines to the oxidative stress generated by photoactivated methylene blue (MB) and KBrO3 treatments. The studied cells are derived from patients with XPD mutations but expressing different clinical phenotypes, including xeroderma pigmentosum (XP), XP and Cockayne syndrome (XP-D/CS) and trichothiodystrophy (TTD). We show by different approaches that all XPD-mutated cell lines tested were sensitive to oxidative stress, with those from TTD patients being the most sensitive. Host cell reactivation (HCR) assays showed that XP-D/CS and TTD cells have severely impaired repair capacity of oxidised lesions in plasmid DNA, and alkaline comet assays demonstrated the induction of significantly higher amounts of DNA strand breaks after treatment with photoactivated MB in these cells compared to wild-type cells. All XPD-mutated cells presented strong S/G2 arrest and persistent γ-H2AX staining after photoactivated MB treatment. Taken together, these results indicate that XPD participates in the repair of lesions induced by the redox process, and that XPD mutations lead to differences in the response to oxidatively induced damage.

Predominant role of DNA polymerase eta and p53-dependent translesion synthesis in the survival of ultraviolet-irradiated human cells.

Genome lesions trigger biological responses that help cells manage damaged DNA, improving cell survival. Pol eta is a translesion synthesis (TLS) polymerase that bypasses lesions that block replicative polymerases, avoiding continued stalling of replication forks, which could lead to cell death. p53 also plays an important role in preventing cell death after ultraviolet (UV) light exposure. Intriguingly, we show that p53 does so by favoring translesion DNA synthesis by pol eta. In fact, the p53-dependent induction of pol eta in normal and DNA repair-deficient XP-C human cells after UV exposure has a protective effect on cell survival after challenging UV exposures, which was absent in p53- and Pol H-silenced cells. Viability increase was associated with improved elongation of nascent DNA, indicating the protective effect was due to more efficient lesion bypass by pol eta. This protection was observed in cells proficient or deficient in nucleotide excision repair, suggesting that, from a cell survival perspective, proper bypass of DNA damage can be as relevant as removal. These results indicate p53 controls the induction of pol eta in DNA damaged human cells, resulting in improved TLS and enhancing cell tolerance to DNA damage, which parallels SOS responses in bacteria.

Overexpression of KLC2 due to a homozygous deletion in the non-coding region causes SPOAN syndrome.

SPOAN syndrome is a neurodegenerative disorder mainly characterized by spastic paraplegia, optic atrophy and neuropathy (SPOAN). Affected patients are wheelchair bound after 15 years old, with progressive joint contractures and spine deformities. SPOAN patients also have sub normal vision secondary to apparently non-progressive congenital optic atrophy. A potential causative gene was mapped at 11q13 ten years ago. Here we performed next-generation sequencing in SPOAN-derived samples. While whole-exome sequencing failed to identify the causative mutation, whole-genome sequencing allowed to detect a homozygous 216-bp deletion (chr11.hg19:g.66,024,557_66,024,773del) located at the non-coding upstream region of the KLC2 gene. Expression assays performed with patient's fibroblasts and motor neurons derived from SPOAN patients showed KLC2 overexpression. Luciferase assay in constructs with 216-bp deletion confirmed the overexpression of gene reporter, varying from 48 to 74%, as compared with wild-type. Knockdown and overexpression of klc2 in Danio rerio revealed mild to severe curly-tail phenotype, which is suggestive of a neuromuscular disorder. Overexpression of a gene caused by a small deletion in the non-coding region is a novel mechanism, which to the best of our knowledge, was never reported before in a recessive condition. Although the molecular mechanism of KLC2 up-regulation still remains to be uncovered, such example adds to the importance of non-coding regions in human pathology.

Mutation in PNKP presenting initially as axonal Charcot-Marie-Tooth disease.

PNKP (polynucleotide kinase 3'-phosphatase, OMIM #605610) product is involved in the repair of strand breaks and base damage in the DNA molecule mainly caused by radical oxygen species. Deleterious variants affecting this gene have been previously associated with microcephaly, epilepsy, and developmental delay.(1) According to a previous report, homozygous loss-of-function substitution in PNKP was associated with cerebellar atrophy, neuropathy, microcephaly, epilepsy, and intellectual disability.(2) Recently, whole-exome sequencing (WES) performed in a cohort of Portuguese families with ataxia with oculomotor apraxia (AOA) disclosed pathogenic variants in PNKP in 11 individuals. Other clinical features in that study included neuropathy, dystonia, cognitive impairment, decreased vibration sense, pyramidal signs, mild elevation in α-fetoprotein, and low levels of albumin. This condition was named AOA type 4 (OMIM #616267), as the phenotype of AOA has been previously associated with 3 other genes: APTX, SETX, and PIK3R5.(3) Altogether, these reports demonstrate the great phenotypic diversity associated with PNKP mutations. In this article, we further enlarge this variability by demonstrating that early-onset axonal sensory-motor neuropathy (or axonal Charcot-Marie-Tooth (CMT) disease) followed years later by ataxia without oculomotor apraxia can be caused by deleterious variants in PNKP. Full consent was obtained from the patient and his parents for this publication. This study was approved by institutional ethics committees.

Gap-filling and bypass at the replication fork are both active mechanisms for tolerance of low-dose ultraviolet-induced DNA damage in the human genome.

Ultraviolet (UV)-induced DNA damage are removed by nucleotide excision repair (NER) or can be tolerated by specialized translesion synthesis (TLS) polymerases, such as Polη. TLS may act at stalled replication forks or through an S-phase independent gap-filling mechanism. After UVC irradiation, Polη-deficient (XP-V) human cells were arrested in early S-phase and exhibited both single-strand DNA (ssDNA) and prolonged replication fork stalling, as detected by DNA fiber assay. In contrast, NER deficiency in XP-C cells caused no apparent defect in S-phase progression despite the accumulation of ssDNA and a G2-phase arrest. These data indicate that while Polη is essential for DNA synthesis at ongoing damaged replication forks, NER deficiency might unmask the involvement of tolerance pathway through a gap-filling mechanism. ATR knock down by siRNA or caffeine addition provoked increased cell death in both XP-V and XP-C cells exposed to low-dose of UVC, underscoring the involvement of ATR/Chk1 pathway in both DNA damage tolerance mechanisms. We generated a unique human cell line deficient in XPC and Polη proteins, which exhibited both S- and G2-phase arrest after UVC irradiation, consistent with both single deficiencies. In these XP-C/Polη(KD) cells, UVC-induced replicative intermediates may collapse into double-strand breaks, leading to cell death. In conclusion, both TLS at stalled replication forks and gap-filling are active mechanisms for the tolerance of UVC-induced DNA damage in human cells and the preference for one or another pathway depends on the cellular genotype.

Novel XPG (ERCC5) mutations affect DNA repair and cell survival after ultraviolet but not oxidative stress.

Nucleotide excision repair (NER) is the most flexible of all known DNA-repair mechanisms, and XPG is a 3'-endonuclease that participates in NER. Mutations in this gene (ERCC5) may result in the human syndrome xeroderma pigmentosum (XP) and, in some cases, in the complex phenotype of Cockayne syndrome (CS). Two Brazilian XP siblings, who were mildly affected, were investigated and classified into the XP-G group. The cells from these patients were highly ultraviolet (UV) sensitive but not sensitive to photosensitized methylene blue, an agent that causes oxidative stress. This phenotype is in contrast to XP-G/CS cells, which are highly sensitive to this oxidative agent. Sequencing revealed a compound heterozygous genotype with two novel missense mutations: c.83C>A (p.Ala28Asp) and c.2904G>C (p.Trp968Cys). The first mutation maps to the catalytic site of the XPG protein, whereas the second may compromise binding to DNA. Functional assays indicated that the mutated alleles were unable to perform the complete repair of UV-irradiated plasmids; however, full correction was observed for oxidatively damaged plasmids. Therefore, the XP phenotype of these patients is caused by novel missense mutations that specifically affect DNA repair for UV- but not oxidative-stress-induced DNA damage, and implications for XP versus XP/CS phenotype are discussed.

Expression of NADPH oxidase by trophoblast cells: potential implications for the postimplanting mouse embryo.

Cytochemical localization of hydrogen peroxide-generating sites suggests NADPH (nicotinamide adenine dinucleotide 3-phosphate [reduced form]) oxidase expression at the maternal-fetal interface. To explore this possibility, we have characterized the expression and activity of the NADPH oxidase complex in trophoblast cells during the postimplantation period. Implantation sites and ectoplacental cones (EPCs) from 7.5-gestational day embryos from CD1 mice were used as a source for expression analyses of NADPH oxidase catalytic and regulatory subunits. EPCs grown in primary culture were used to investigate the production of superoxide anion through dihydroxyethidium oxidation in confocal microscopy and immunohistochemical assays. NADPH subunits Cybb (gp91phox), Cyba (p22phox), Ncf4 (p40phox), Ncf1 (p47phox), Ncf2 (p67phox), and Rac1 were expressed by trophoblast cells. The fundamental subunits of membrane CYBB and cytosolic NCF2 were markedly upregulated after phorbol-12-myristate-13-acetate (PMA) treatment, as detected by quantitative real-time PCR, Western blotting, and immunohistochemistry. Fluorescence microscopy imaging showed colocalization of cytosolic and plasma membrane NADPH oxidase subunits mainly after PMA treatment, suggesting assembly of the complex after enzyme activation. Cultured EPCs produced superoxide in a NADPH-dependent manner, associating the NADPH oxidase-mediated superoxide production with postimplantation trophoblast physiology. NADPH-oxidase cDNA subunit sequencing showed a high degree of homology between the trophoblast and neutrophil isoforms of the oxidase, emphasizing a putative role for reactive oxygen species production in phagocytic activity and innate immune responses.