Endogenous and exogenous agents that can damage DNA are a constant threat to genome stability in all living cells. In response, cells have evolved an array of mechanisms to repair DNA damage or to eliminate the cells damaged beyond repair. One of these mechanisms is nucleotide excision repair (NER) which is the major repair pathway responsible for removing a wide variety of bulky DNA lesions. Deficiency, or mutation, in one or several of the NER repair proteins is responsible for many diseases, including cancer. Prokaryotic NER involves only three proteins to recognize and incise a damaged site, while eukaryotic NER requires more than 25 proteins to efficiently recognize and incise a damaged site. XPC-RAD23B (XPC) is the damage recognition factor in eukaryotic global genome NER. The association rate of XPC to damaged DNA has been extensively studied; however, our data suggests that the dissociation of the XPC-DNA complex is the rate-limiting step in NER. The factor that verifies DNA-damage downstream of XPC is XPA. XPA also has been implicated in binding of ds-ssDNA junctions and has been found to bind at or near double-strand break sites in the premature aging syndrome Hutchinson-Gilford progeria (HGPS). This role for XPA is outside of its known function in NER and suggests that XPA may bind at collapsed replication forks in HGPS that are unprotected due to a lack of binding by replication proteins. Along with XPC and XPA, ataxia telangiectasia and Rad3-related (ATR) is activated in response to DNA damage and initiates the cell cycle checkpoint pathway to rescue cells from genomic instability. We found that ATR functions outside of its known role in the checkpoint signaling cascade. Our data demonstrate that ATR can rescue cells from apoptosis by inhibiting cytochrome c release at the mitochondria though direct interaction with the outer mitochondrial membrane and the proapoptotic protein tBid. The role of ATR in apoptosis is regulated by Pin1, which can change the structure of ATR at the backbone level. All of the results presented here suggest novel roles for DNA repair proteins in the maintenance of genome stability.

Melanoma is one of the most aggressive types of cancer. It originates from the transformation of melanocytes present in the epidermal/dermal junction of the human skin. It is commonly accepted that melanomagenesis is influenced by the interaction of environmental factors, genetic factors, as well as tumor-host interactions. DNA photoproducts induced by UV radiation are, in normal cells, repaired by the nucleotide excision repair (NER) pathway. The prominent role of NER in cancer resistance is well exemplified by patients with Xeroderma Pigmentosum (XP). This disease results from mutations in the components of the NER pathway, such as XPA and XPC proteins. In humans, NER pathway disruption leads to the development of skin cancers, including melanoma. Similar to humans afflicted with XP, Xpa and Xpc deficient mice show high sensibility to UV light, leading to skin cancer development, except melanoma. The Endothelin 3 (Edn3) signaling pathway is essential for proliferation, survival and migration of melanocyte precursor cells. Excessive production of Edn3 leads to the accumulation of large numbers of melanocytes in the mouse skin, where they are not normally found. In humans, Edn3 signaling pathway has also been implicated in melanoma progression and its metastatic potential. The goal of this study was the development of the first UV-induced melanoma mouse model dependent on the over-expression of Edn3 in the skin. The UV-induced melanoma mouse model reported here is distinguishable from all previous published models by two features: melanocytes are not transformed a priori and melanomagenesis arises only upon neonatal UV exposure. In this model, melanomagenesis depends on the presence of Edn3 in the skin. Disruption of the NER pathway due to the lack of Xpa or Xpc proteins was not essential for melanomagenesis; however, it enhanced melanoma penetrance and decreased melanoma latency after one single neonatal erythemal UV dose. Exposure to a second dose of UV at six weeks of age did not change time of appearance or penetrance of melanomas in this mouse model. Thus, a combination of neonatal UV exposure with excessive Edn3 in the tumor microenvironment is sufficient for melanomagenesis in mice; furthermore, NER deficiency exacerbates this process.

No description available.

A common feature of progeria syndromes is a premature aging phenotype and an enhanced accumulation of DNA damage arising from a compromised repair system. HGPS (Hutchinson-Gilford progeria syndrome) is a severe form of progeria in which patients accumulate progerin, a mutant lamin A protein derived from a splicing variant of the lamin A/C gene (LMNA). Progerin causes chromatin perturbations which result in the formation of DSBs (double-strand breaks) and abnormal DDR (DNA-damage response). In the present article, we review recent findings which resolve some mechanistic details of how progerin may disrupt DDR pathways in HGPS cells. We propose that progerin accumulation results in disruption of functions of some replication and repair factors, causing the mislocalization of XPA (xeroderma pigmentosum group A) protein to the replication forks, replication fork stalling and, subsequently, DNA DSBs. The binding of XPA to the stalled forks excludes normal binding by repair proteins, leading to DSB accumulation, which activates ATM (ataxia telangiectasia mutated) and ATR (ATM- and Rad3-related) checkpoints, and arresting cell-cycle progression.

No description available.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-04-17T23:12:49Z No. of bitstreams: 1 AngelicaMariaDeSousaLeal_TESE.pdf: 6582579 bytes, checksum: 5f557c13b6008a7677f62167674670fe (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-04-20T22:14:08Z (GMT) No. of bitstreams: 1 AngelicaMariaDeSousaLeal_TESE.pdf: 6582579 bytes, checksum: 5f557c13b6008a7677f62167674670fe (MD5) / Made available in DSpace on 2017-04-20T22:14:08Z (GMT). No. of bitstreams: 1 AngelicaMariaDeSousaLeal_TESE.pdf: 6582579 bytes, checksum: 5f557c13b6008a7677f62167674670fe (MD5) Previous issue date: 2016-09-29 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / No contexto da resposta ao estresse oxidativo, o reparo por excis?o de bases (BER) ? considerado a principal via para o reparo de les?es oxidadas. Entretanto, estudos indicam o papel do reparo por excis?o de nucleot?deos (NER) na corre??o dessas les?es. Al?m disso, fatores do NER j? tiveram fun??es descritas em outros processos biol?gicos, sendo importante que se busque novas fun??es biol?gicas que possam ser associadas aos fen?tipos das s?ndromes causadas por muta??es nos genes da via NER, dentre elas a Xeroderma pigmentoso grupo de complementa??o A, associada a muta??es em XPA, al?m da s?ndrome de Cockayne, ocasionada por muta??es no gene CSB. Nesse contexto, c?lulas deficientes em XPA (XP12RO) ou CSB (CS1AN) foram submetidas ao estresse oxidativo com per?xido de hidrog?nio (H2O2) e apresentaram um perfil de sensibilidade ao agente, indicando que a aus?ncia dessas prote?nas sensibilizou as linhagens a essa condi??o. A an?lise do transcriptoma de c?lulas XP12RO indicou a diminui??o na express?o de genes com papel na resposta ao dano no DNA e que promovem a sobreviv?ncia celular em resposta ao estresse oxidativo. Nesse cen?rio, os resultados indicaram que XPA pode atuar na regula??o da express?o de genes essenciais ? resposta ao dano no DNA e na sobreviv?ncia ao estresse oxidativo (EGR1, GADD45A, GADD45B e XPC). Por outro lado, a an?lise do transcriptoma de c?lulas CS1AN indicaram a diminui??o na express?o de genes-chave nos processos biol?gicos como transcri??o, processamento de mRNA, prote?lise via ubiquitina-proteassoma ou respira??o celular, indicando um poss?vel papel central da prote?na CSB na regula??o desses processos, em resposta ao estresse oxidativo. Al?m disso, dado o fen?tipo de neurodegenera??o associada a s?ndrome de Cockayne, c?lulas progenitoras neurais (NPCs) e neur?nios derivados de c?lulas-tronco pluripotentes induzidas (iPSCs) deficientes em CSB foram utilizados como modelos de estudo de altera??es neuronais in vitro, de modo que os resultados indicaram que assim como observado nos fibroblastos, c?lulas NPCs deficientes em CSB tamb?m apresentaram sensibilidade a agentes oxidantes. Ainda, os resultados mostraram que assim como observado no transcriptoma de fibroblastos CS1AN, dada a diminui??o na express?o de genes com papel na respira??o celular, as an?lises do consumo de oxig?nio em neur?nios deficientes em CSB indicaram uma poss?vel disfun??o mitocondrial, caracterizada pelo decr?scimo na taxa de consumo de oxig?nio basal e pela diminui??o das capacidades respirat?rias m?xima ou de reserva dessas c?lulas, sugerindo o papel de CSB no metabolismo mitocondrial em ambos os modelos celulares utilizados neste estudo. / In oxidative stress response, the base excision repair (BER) is considered the major pathway for repair of oxidative lesions. However, an increasing number of studies have indicated the role of nucleotide excision (NER) in the repair of these lesions. In addition, some NER factors had functions beyond the role in repair already described and it is important to search for new molecular functions that can be associated to the classical phenotypes of the syndromes caused by mutations in NER genes: Xeroderma pigmentosum complementation group A, caused by mutations in XPA and Cockayne syndrome, caused by mutations in CSB. In this context, XPA (XP12RO) or CSB (CS1AN) deficient cells were submitted to oxidative stress induced by Hydrogen peroxide (H2O2) and the results indicated that both cell lines showed sensitivity to this agent. Furthermore, the transcriptome of XP12RO cells revealed the downregulation of genes that play a role in DNA damage response and promote cell survival in response to oxidative stress. In this scenario, the results indicated that XPA regulates the expression of genes that play a key role in DNA damage response and promote survival in response to stress (EGR1, GADD45A, GADD45B and XPC). On the other hand, the transcriptome analysis of CS1AN cells showed the downregulation of genes that play a key role in biological processes such as transcription, mRNA processing, protein degradation by the ubiquitin?proteasome pathway proteolysis or cellular respiration, indicating a possible role for CSB protein in the regulation of these processes, in response to oxidative stress. In adittion, given the neurodegeneration phenotype associated to Cockayne syndrome, neural progenitor cells (NPCs) and neurons derived from CSB deficient induced pluripotent stem cells (iPSCs) were used as cellular models to analyse neuronal changes in vitro. The results showed that, as observed in fibroblasts CS1AN, NPCs also presented sensitivity to oxidizing agents. Furthermore, as indicated in the transcriptome data from CS1AN fibroblasts, given the downregulation of genes that play a pivotal role in cellular respiration, the analysis of oxygen consumption rates in CSB deficient neurons also indicated a mitochondrial dysfunction characterized by the decrease in oxygen consumption basal rate and a lower maximum respiratory and reserve capacities, suggesting that the lack of functional CSB leads to a mitochondrial dysfunction in both cellular models used in this study. / 2017-12-09