RecQ helicase expression in patients with telomeropathies.

BACKGROUND: Telomeropathies are a group of inherited disorders caused by germline pathogenic variants in genes involved in telomere maintenance, resulting in excessive telomere attrition that affects several tissues, including hematopoiesis. RecQ and RTEL1 helicases contribute to telomere maintenance by unwinding telomeric structures such as G-quadruplexes (G4), preventing replication defects. Germline RTEL1 variants also are etiologic in telomeropathies. METHODS AND RESULTS: Here we investigated the expression of RecQ (RECQL1, BLM, WRN, RECQL4, and RECQL5) and RTEL1 helicase genes in peripheral blood mononuclear cells (PBMCs) from human telomeropathy patients. The mRNA expression levels of all RecQ helicases, but not RTEL1, were significantly downregulated in patients' primary cells. Reduced RecQ expression was not attributable to cell proliferative exhaustion, as RecQ helicases were not attenuated in T cells exhausted in vitro. An additional fifteen genes involved in DNA damage repair and RecQ functional partners also were downregulated in the telomeropathy cells. CONCLUSION: These findings indicate that the expression of RecQ helicases and functional partners involved in DNA repair is downregulated in PBMCs of telomeropathy patients.

Mutational signatures and increased retrotransposon insertions in xeroderma pigmentosum variant skin tumors.

Xeroderma pigmentosum variant (XP-V) is an autosomal recessive disease with an increased risk of developing cutaneous neoplasms in sunlight-exposed regions. These cells are deficient in the translesion synthesis (TLS) DNA polymerase eta, responsible for bypassing different types of DNA lesions. From the exome sequencing of 11 skin tumors of a genetic XP-V patients' cluster, classical mutational signatures related to sunlight exposure, such as C>T transitions targeted to pyrimidine dimers, were identified. However, basal cell carcinomas also showed distinct C>A mutation spectra reflecting a mutational signature possibly related to sunlight-induced oxidative stress. Moreover, four samples carry different mutational signatures, with C>A mutations associated with tobacco chewing or smoking usage. Thus, XP-V patients should be warned of the risk of these habits. Surprisingly, higher levels of retrotransposon somatic insertions were also detected when the tumors were compared with non-XP skin tumors, revealing other possible causes for XP-V tumors and novel functions for the TLS polymerase eta in suppressing retrotransposition. Finally, the expected high mutation burden found in most of these tumors renders these XP patients good candidates for checkpoint blockade immunotherapy.

DNA damage and oxidative stress in human cells infected by Trypanosoma cruzi.

Trypanosoma cruzi is the etiologic agent of Chagas' disease. Infected cells with T. cruzi activate several responses that promote unbalance of reactive oxygen species (ROS) that may cause DNA damage that activate cellular responses including DNA repair processes. In this work, HeLa cells and AC16 human cardiomyocyte cell line were infected with T. cruzi to investigate host cell responses at genome level during parasites intracellular life cycle. In fact, alkaline sensitive sites and oxidized DNA bases were detected in the host cell genetic material particularly in early stages of infection. These DNA lesions were accompanied by phosphorylation of the histone H2Ax, inducing γH2Ax, a marker of genotoxic stress. Moreover, Poly [ADP-ribose] polymerase-1 (PARP1) and 8-oxoguanine glycosylase (OGG1) are recruited to host cell nuclei, indicating activation of the DNA repair process. In infected cells, chromatin-associated proteins are carbonylated, as a possible consequence of oxidative stress and the nuclear factor erythroid 2-related factor 2 (NRF2) is induced early after infection, suggesting that the host cell antioxidant defenses are activated. However, at late stages of infection, NRF2 is downregulated. Interestingly, host cells treated with glutathione precursor, N-acetyl cysteine, NRF2 activator (Sulforaphane), and also Benznidonazol (BNZ) reduce parasite burst significantly, and DNA damage. These data indicate that the balance of oxidative stress and DNA damage induction in host cells may play a role during the process of infection itself, and interference in these processes may hamper T. cruzi infection, revealing potential target pathways for the therapy support.

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.

Cockayne syndrome-derived neurons display reduced synapse density and altered neural network synchrony.

Cockayne syndrome (CS) is a rare genetic disorder in which 80% of cases are caused by mutations in the Excision Repair Cross-Complementation group 6 gene (ERCC6). The encoded ERCC6 protein is more commonly referred to as Cockayne Syndrome B protein (CSB). Classical symptoms of CS patients include failure to thrive and a severe neuropathology characterized by microcephaly, hypomyelination, calcification and neuronal loss. Modeling the neurological aspect of this disease has proven difficult since murine models fail to mirror classical neurological symptoms. Therefore, a robust human in vitro cellular model would advance our fundamental understanding of the disease and reveal potential therapeutic targets. Herein, we successfully derived functional CS neural networks from human CS induced pluripotent stem cells (iPSCs) providing a new tool to facilitate studying this devastating disease. We identified dysregulation of the Growth Hormone/Insulin-like Growth Factor-1 (GH/IGF-1) pathway as well as pathways related to synapse formation, maintenance and neuronal differentiation in CSB neurons using unbiased RNA-seq gene expression analyses. Moreover, when compared to unaffected controls, CSB-deficient neural networks displayed altered electrophysiological activity, including decreased synchrony, and reduced synapse density. Collectively, our work reveals that CSB is required for normal neuronal function and we have established an alternative to previously available models to further study neural-specific aspects of CS.

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.

ATR suppresses apoptosis after UVB irradiation by controlling both translesion synthesis and alternative tolerance pathways.

Ultraviolet (UV) light can stall replication forks owing to the formation of bulky lesions in the DNA. Replication across these blocking lesions occurs through translesion DNA synthesis, and cells activate the ATR damage responses to UV. However, it remains unclear whether lesion bypass requires the replication checkpoint because ATR is not necessary for PCNA ubiquitylation. We observed that ATR knockdown by siRNA increased replication stress and promoted early induction of apoptosis following UVB irradiation in SV40-immortalized human cells, including cells from XP-V and XP-C patients. XP-V cells were further sensitized by the silencing, indicating that DNA polymerase η (Pol η) remains active despite ATR control. However, following UVB irradiation, ATR-depleted cells were unable to achieve mitosis, as would be expected after the loss of a DNA checkpoint control. Thus, ATR also regulates replication arrest recovery following UVB-induced damage, independently of Pol η, in SV40-immortalized cell lines. The ATR-mediated DNA damage response regulates replication and different tolerance pathways, and in these cells, ATR depletion induces replication catastrophe, which contributes to explain the potential of ATR inhibition to protect against UVB-induced carcinogenesis.

DNA repair and recovery of RNA synthesis following exposure to ultraviolet light are delayed in long genes.

The kinetics of DNA repair and RNA synthesis recovery in human cells following UV-irradiation were assessed using nascent RNA Bru-seq and quantitative long PCR. It was found that UV light inhibited transcription elongation and that recovery of RNA synthesis occurred as a wave in the 5'-3' direction with slow recovery and TC-NER at the 3' end of long genes. RNA synthesis resumed fully at the 3'-end of genes after a 24 h recovery in wild-type fibroblasts, but not in cells deficient in transcription-coupled nucleotide excision repair (TC-NER) or global genomic NER (GG-NER). Different transcription recovery profiles were found for individual genes but these differences did not fully correlate to differences in DNA repair of these genes. Our study gives the first genome-wide view of how UV-induced lesions affect transcription and how the recovery of RNA synthesis of large genes are particularly delayed by the apparent lack of resumption of transcription by arrested polymerases.

Autophagy Roles in the Modulation of DNA Repair Pathways.

Autophagy and DNA repair are biological processes vital for cellular homeostasis maintenance and when dysfunctional, they lead to several human disorders including premature aging, neurodegenerative diseases, and cancer. The interchange between these pathways is complex and it may occur in both directions. Autophagy is activated in response to several DNA lesions types and it can regulate different mechanisms and molecules involved in DNA damage response (DDR), such as cell cycle checkpoints, cell death, and DNA repair. Thus, autophagy may modulate DNA repair pathways, the main focus of this review. In addition to the already well-documented autophagy positive effects on homologous recombination (HR), autophagy has also been implicated with other DNA repair mechanisms, such as base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR). Given the relevance of these cellular processes, the clinical applications of drugs targeting this autophagy-DNA repair interface emerge as potential therapeutic strategies for many diseases, especially cancer.

Microenvironment and autophagy cross-talk: Implications in cancer therapy.

There are many ongoing clinical trials to validate tumour microenvironment or autophagic pathway components as targets for anticancer therapies. Different components of the tumour microenvironment play important roles in tumour cell responses, directly affecting malignant transformation, drug resistance and metastasis. Autophagy is also related to chemotherapy responses by inducing tumour cell death or survival. Thus, the autophagy pathway may act as oncosuppressor, in addition to protecting cells from chemotherapy. The cross-talk between the microenvironment and autophagy is very complex and poorly understood. In a recent study using a three-dimensional (3D) cell culture model, the well-documented chemotherapy-mediated activation of autophagy was impaired in breast cancer cells, suggesting a context-dependent outcome for autophagy modulators, under the control of the p53 protein. A deeper understanding of this microenvironment/autophagy interplay may provide important clues for identifying differences in the tumour cell signalling network from in vitro basic research studies to the actual clinical context. In this work, we summarize the role of the microenvironment and autophagy in physiological and tumourigenic conditions, their interactions, and the challenges related to the use of drugs that target these pathways in cancer treatment protocols, emphasizing the potential use of 3D cell culture models in preclinical studies.

The intronic long noncoding RNA ANRASSF1 recruits PRC2 to the RASSF1A promoter, reducing the expression of RASSF1A and increasing cell proliferation.

The down-regulation of the tumor-suppressor gene RASSF1A has been shown to increase cell proliferation in several tumors. RASSF1A expression is regulated through epigenetic events involving the polycomb repressive complex 2 (PRC2); however, the molecular mechanisms modulating the recruitment of this epigenetic modifier to the RASSF1 locus remain largely unknown. Here, we identify and characterize ANRASSF1, an endogenous unspliced long noncoding RNA (lncRNA) that is transcribed from the opposite strand on the RASSF1 gene locus in several cell lines and tissues and binds PRC2. ANRASSF1 is transcribed through RNA polymerase II and is 5'-capped and polyadenylated; it exhibits nuclear localization and has a shorter half-life compared with other lncRNAs that bind PRC2. ANRASSF1 endogenous expression is higher in breast and prostate tumor cell lines compared with non-tumor, and an opposite pattern is observed for RASSF1A. ANRASSF1 ectopic overexpression reduces RASSF1A abundance and increases the proliferation of HeLa cells, whereas ANRASSF1 silencing causes the opposite effects. These changes in ANRASSF1 levels do not affect the RASSF1C isoform abundance. ANRASSF1 overexpression causes a marked increase in both PRC2 occupancy and histone H3K27me3 repressive marks, specifically at the RASSF1A promoter region. No effect of ANRASSF1 overexpression was detected on PRC2 occupancy and histone H3K27me3 at the promoter regions of RASSF1C and the four other neighboring genes, including two well-characterized tumor suppressor genes. Additionally, we demonstrated that ANRASSF1 forms an RNA/DNA hybrid and recruits PRC2 to the RASSF1A promoter. Together, these results demonstrate a novel mechanism of epigenetic repression of the RASSF1A tumor suppressor gene involving antisense unspliced lncRNA, in which ANRASSF1 selectively represses the expression of the RASSF1 isoform overlapping the antisense transcript in a location-specific manner. In a broader perspective, our findings suggest that other non-characterized unspliced intronic lncRNAs transcribed in the human genome might contribute to a location-specific epigenetic modulation of genes.

Highly sensitive biological assay for determining the photoprotective efficacy of sunscreen.

The protective effect of sunscreens has been extensively evaluated in vivo as a measure of erythema induced in human skin and is expressed as Sun Protection Factor (SPF). In vitro alternatives that use human cells might overcome the limitations of testing on human beings. Here is proposed a broad and accurate in vitro approach for evaluating the efficacy of commercial sunscreens even under environmental conditions. This Cell dosimeter allowed the determination of Sun Protection Factor for DNA (DNA-SPF), using specific DNA repair enzymes and antibodies, and Sun Protection Factor for Lethal Damage (LD-SPF), by measuring cell viability and apoptosis induced after the irradiation of human cells. The use of xeroderma pigmentosum (XP) cells, which are deficient in DNA repair, rendered this assay more sensitive. The results revealed significant protection against the effects elicited by UVB radiation; however, there was no efficient protection from DNA lesions and cell death induced by UVA radiation or natural sunlight. This work demonstrates the environmental application of this biodosimeter for measuring UV-induced biological damage to human cells and supports the need for better evaluation of the UVA protection efficacy conferred by commercial sunscreens, in terms of induction of DNA lesions and cell death.

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.

Transcriptome analysis of the acoelomate human parasite Schistosoma mansoni.

Schistosoma mansoni is the primary causative agent of schistosomiasis, which affects 200 million individuals in 74 countries. We generated 163,000 expressed-sequence tags (ESTs) from normalized cDNA libraries from six selected developmental stages of the parasite, resulting in 31,000 assembled sequences and 92% sampling of an estimated 14,000 gene complement. By analyzing automated Gene Ontology assignments, we provide a detailed view of important S. mansoni biological systems, including characterization of metazoa-specific and eukarya-conserved genes. Phylogenetic analysis suggests an early divergence from other metazoa. The data set provides insights into the molecular mechanisms of tissue organization, development, signaling, sexual dimorphism, host interactions and immune evasion and identifies novel proteins to be investigated as vaccine candidates and potential drug targets.

Genomic mutation landscape of skin cancers from DNA repair-deficient xeroderma pigmentosum patients.

Xeroderma pigmentosum (XP) is a genetic disorder caused by mutations in genes of the Nucleotide Excision Repair (NER) pathway (groups A-G) or in Translesion Synthesis DNA polymerase η (V). XP is associated with an increased skin cancer risk, reaching, for some groups, several thousand-fold compared to the general population. Here, we analyze 38 skin cancer genomes from five XP groups. We find that the activity of NER determines heterogeneity of the mutation rates across skin cancer genomes and that transcription-coupled NER extends beyond the gene boundaries reducing the intergenic mutation rate. Mutational profile in XP-V tumors and experiments with POLH knockout cell line reveal the role of polymerase η in the error-free bypass of (i) rare TpG and TpA DNA lesions, (ii) 3' nucleotides in pyrimidine dimers, and (iii) TpT photodimers. Our study unravels the genetic basis of skin cancer risk in XP and provides insights into the mechanisms reducing UV-induced mutagenesis in the general population.

Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay.

The DNA fiber assay is a simple and robust method for the analysis of replication fork dynamics, based on the immunodetection of nucleotide analogs that are incorporated during DNA synthesis in human cells. However, this technique has a limited resolution of a few thousand kilobases. Consequently, post-replicative single-stranded DNA (ssDNA) gaps as small as a few hundred bases are not detectable by the standard assay. Here, we describe a modified version of the DNA fiber assay that utilizes the S1 nuclease, an enzyme that specifically cleaves ssDNA. In the presence of post-replicative ssDNA gaps, the S1 nuclease will target and cleave the gaps, generating shorter tracts that can be used as a read-out for ssDNA gaps on ongoing forks. These post-replicative ssDNA gaps are formed when damaged DNA is replicated discontinuously. They can be repaired via mechanisms uncoupled from genome replication, in a process known as gap-filling or post-replicative repair. Because gap-filling mechanisms involve DNA synthesis independent of the S phase, alterations in the DNA fiber labeling scheme can also be employed to monitor gap-filling events. Altogether, these modifications of the DNA fiber assay are powerful strategies to understand how post-replicative gaps are formed and filled in the genome of human cells.