Nucleotide excision repair (NER) is one of the major repair systems for removal of DNA lesions. The NER pathway has evolved mainly to repair UV-induced DNA damage and is also active against a broad range of endogenously generated oxidative lesions. Defects in NER result in the human inherited disorder xeroderma pigmentosum (XP), which is characterised by UV hypersensitivity and a 1000-fold increased risk of skin cancer. ERCC1 is essential for the NER pathway where it acts in a complex with the XPF protein to make the incision 5' to the DNA lesion. The normal 1.1kb Ercc1 transcript is expressed in all tissues. Our group has discovered a second larger 1.5 kb transcript, which initiates from an alternative promoter, and is the most abundant Ercc1 transcript in mouse skin. The aims of this project were: 1, To investigate the role of ERCC1 and of the 1.5kb skin specific Ercc1 transcript in protecting the skin against UV-induced DNA damage. 2, To study the importance of ERCC1 in melanoma skin cancer and investigate ERCC1as a possible target for therapy against melanoma. Using a panel of Ercc1 wild-type and deficient cells, we established a quantitative western blotting system to study the expression of ERCC1 in a range of mouse tissues and mouse and human cell types. Although the skin-specific Ercc1 transcript was found to be present at much higher levels in the skin of albino compared to pigmented mouse strains, this did not result in an elevated level of ERCC1 protein. We were also unable to demonstrate that UV-irradiation, or other stress-inducing treatments resulted in increased levels of ERCC1 protein in cultured mouse keratinocytes. We investigated the DNA methylation status of the normal Ercc1 promoter and that of two potential upstream promoter regions that were candidates for the source of the 1.5kb skin-specific Ercc1 transcript. We found no evidence that they were the source and, instead, used 5' RACE analysis to locate the skin-specific promoter to a polymorphic region 500bp upstream of the normal initiation site. In albino strains this region contains a SINE element, which we hypothesize could be involved in the production of the skin-specific Ercc1 transcript. We also investigated the protein level of ERCC1 and other DNA repair proteins, including XPF, MSH2, MSH6 and MLH1 in human melanoma cells and ovarian tumour cells. Significantly elevated protein levels of ERCC1 and XPF, as well as the mismatch repair protein MLH1 were found in melanoma cells. This could possibly contribute to the higher resistance to chemotherapy in melanoma, although the melanoma cell lines we tested did not show increased resistance to UV and cisplatin compared to the ovarian cancer cells tested. When Ercc1 proficient mouse melanoma cells were xenografted into nude mice the xenografts grew rapidly. Cisplatin treatment caused an initial shrinkage of the tumours, but re-growth rapidly followed. Cells re-isolated into culture from cisplatin treated xenografts had significantly higher levels of ERCC1 protein than either input cells, or cells re-isolated from untreated xenografts. An isogenic Ercc1 deficient derivative of the Ercc1 proficient mouse melanoma cell line grew as rapidly as the parent line in vitro, but grew much more slowly as xenografts. In addition, the xenografts shrank completely following cisplatin treatment and did not recover. This suggests that ERCC1 could be a drug target for melanoma therapy.

DNA damage repair is an essential and complex cellular process. Although the basic mechanisms of nucleotide excision repair (NER) have been studied for decades, some mechanistic details remain elusive. The lesion detection step remains one of the most elusive in the process of NER in the contest of chromatin. The work described herein addresses the initial events in the lesion detection step of chromatin repair, also referred to as global genome repair (GG-NER). Both the role of post-translational modifications of lesion identification proteins, and the initial sequence of events in recruitment of repair and remodeling factors are investigated. First, the controversial role of ubiquitination of DDB2 (a human lesion detection protein) is investigated. Due to documented DDB2 function in alternative physiological processes, its direct role in GG-NER is hard to study in human cells. To overcome this obstacle, we established the budding yeast, Saccharomyces cerevisiae as an alternative, simplified model organism to study DDB2-mediated GG-NER. Using this system, we show that inconsistent with the widely accepted model, rapid degradation of DDB2 post-UV irradiation is not an absolute requirement for progression of GG-NER. However, interestingly, our data suggest a role for ubiquitination in the release of DDB2 from chromatin. In both UV and mock treated samples, ubiquitin deficient cells had significantly higher amounts of DDB2 remaining bound to the chromatin compared to the isogenic parent cells. The discussion focuses on the possible physiological relevance of these observations. Additionally, the recruitment of the SWI/SNF chromatin remodeling complex to the silent HML (Hidden MAT Left) locus was also investigated. SWI/SNF is known to require recruitment for its role in transcription; therefore we investigate this requirement in GG-NER. Based on previously published data that indicate an UV-stimulated association of SWI/SNF and Rad4 (a lesion detection protein), we hypothesized that Rad4 is involved in recruitment of SWI/SNF to damaged DNA. Interestingly, our data suggest that Rad4 is not an absolute requirement for recruitment of Snf6 to the HML locus following UV irradiation. However, Rad16 appears to be. These data present an interesting insight into the lesion detection step in GG-NER and this will be discussed.

No description available.

Nucleotide excision repair (NER) and ataxia telangiectasia mutated (ATM)/ATR (ATM- and RAD3-related) NA damage checkpoints are among the major pathways that affect the chemotherapeutic efficiency of the anticancer rug cisplatin. Xeroderma pigmentosum group A (XPA) protein plays a crucial role in NER including both global enome repair (GG-NER) and transcription-coupled repair (TC-NER) subpathways, and has been a potential target for mproving cisplatin therapeutic effects. We report here that XPA translocates from the cytosol into the nucleus after NA damage induced by UV irradiation and cisplatin, a mimetic of UV damage, in human cells with or without p53 deficiency. However, the damage-induced response of XPA nuclear import was significantly slower in p53-deficient cells than in p53-proficient cells. We also found that while XPA is imported into the nucleus upon cisplatin or UV damage in an ATR-dependent manner in p53-proficient A549 lung cancer cells, the ATR checkpoint pathway has no effect on the XPA nuclear import in p53-deficient H1299 lung cancer cells. Similarly, the XPA nuclear translocation is not regulated by ATM checkpoint or by p38MAPK/MK2 either. Our findings suggest that NER is independent on the major DNA damage checkpoint pathways in H1299 (p53-/-) cells and that DNA damage responses are mechanistically different between p53-proficient and p53-deficient cells. Our results also highlight the possibility of selectively targeting XPA nuclear import as a way to sensitize cisplatin anticancer activity, but targeting ATR/ATM-dependent checkpoints may not be helpful in killing p53-deficient cancer cells.

Single cell gel electrophoresis (SCGE), more commonly known as the Comet assay, is an uncomplicated, affordable and versatile method for investigating DNA damage and repair. Existing comet–assay based methods were modified and applied in this study in order to examine the effects of different perturbations on the DNA repair capacity of different samples. Mitochondrial functioning has a vast effect on overall cell physiology and does not simply involve the production of energy in the form of ATP that sustains common biological processes, but is also associated with important cellular occurrences such as apoptosis and ROS production. It is suggested that a change in mitochondrial function may lead to extensive ROS production which may negatively affect macromolecules, including proteins involved in DNA repair pathways, and impaired energy formation which in turn may hamper the proper occurrence of energy driven processes. Complex I and ?III knock–down systems established in 143B cells are used to investigate the effect that perturbations of the energy metabolism may have on DNA repair capacity. Metallothioneins (MTs) are known to play an imperative role in trace element homeostasis and detoxification of metals and are effective ROS scavengers. The prooxidant environment that heavy metal imbalance causes may result in mutagenesis and transformation through DNA damage. It is suggested that an imbalance in the metal homeostasis caused by MT knock–out may create an environment favourable for DNA damage formation and at the same time impair DNA repair pathways. Because of the multi–functionality and involvement of metallothioneins in such a wide variety of biological processes, it was considered interesting and essential to extend the investigation on the effect of the absence of metallothioneins on DNA repair. A metallothionein I and ?II knock–out mouse model is employed to determine the effect of MT knock–out on DNA repair capacity. It was clear from the results obtained that transfection of cells, as used to investigate a perturbation in the energy metabolism in 143B cells, has an impairing effect on DRC. It was also confirmed that metallothioneins play an important and diverse role in cell biology since the absence thereof inhibits both BER and NER. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2011.

Single cell gel electrophoresis (SCGE), more commonly known as the Comet assay, is an uncomplicated, affordable and versatile method for investigating DNA damage and repair. Existing comet–assay based methods were modified and applied in this study in order to examine the effects of different perturbations on the DNA repair capacity of different samples. Mitochondrial functioning has a vast effect on overall cell physiology and does not simply involve the production of energy in the form of ATP that sustains common biological processes, but is also associated with important cellular occurrences such as apoptosis and ROS production. It is suggested that a change in mitochondrial function may lead to extensive ROS production which may negatively affect macromolecules, including proteins involved in DNA repair pathways, and impaired energy formation which in turn may hamper the proper occurrence of energy driven processes. Complex I and ?III knock–down systems established in 143B cells are used to investigate the effect that perturbations of the energy metabolism may have on DNA repair capacity. Metallothioneins (MTs) are known to play an imperative role in trace element homeostasis and detoxification of metals and are effective ROS scavengers. The prooxidant environment that heavy metal imbalance causes may result in mutagenesis and transformation through DNA damage. It is suggested that an imbalance in the metal homeostasis caused by MT knock–out may create an environment favourable for DNA damage formation and at the same time impair DNA repair pathways. Because of the multi–functionality and involvement of metallothioneins in such a wide variety of biological processes, it was considered interesting and essential to extend the investigation on the effect of the absence of metallothioneins on DNA repair. A metallothionein I and ?II knock–out mouse model is employed to determine the effect of MT knock–out on DNA repair capacity. It was clear from the results obtained that transfection of cells, as used to investigate a perturbation in the energy metabolism in 143B cells, has an impairing effect on DRC. It was also confirmed that metallothioneins play an important and diverse role in cell biology since the absence thereof inhibits both BER and NER. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2011.

O câncer é a primeira causa de mortes por doença, após 1 ano de idade, até o final da adolescência, excetuando aquelas relacionadas aos acidentes e à violência. A Leucemia Linfoide Aguda (LLA) afeta células linfoides e agrava-se rapidamente. São os tumores mais frequentes na infância e representam um terço de todas as neoplasias malignas nesta faixa etária. Em média, a taxa de cura excede 70%, todavia, apesar dos avanços das últimas décadas, os índices de crianças que apresentam recidiva da doença continua significativo. Danos endógenos ao DNA ocorrem numa frequência altíssima, além dos danos causados por terapias antitumorais. Alteração no reparo ao dano do DNA pode induzir mecanismos de resistência ao tratamento quimioterápico, resultando em aumento do reparo de lesões do DNA. Reparo por Excisão de Nucleotídeos (NER) é a via de reparo de DNA mais versátil e flexível nas células. Seus componentes estão sendo estudados como biomarcadores de prognóstico e terapias-alvo. No entanto, alguns relatórios têm abordado danos de DNA em Leucemia Linfoide Aguda (LLA) pediátrica. Neste estudo, realizamos um estudo de acompanhamento observacional em pacientes pediátricos para avaliar os danos do DNA pelo Ensaio Cometa Alcalino e expressão gênica da via de NER durante a indução da quimioterapia. Amostras de medula óssea (MO) ao diagnóstico, dia 15 (D15) e 30 (D30) do tratamento foram coletadas de 28 pacientes com LLA. Não houve aumento no índice de dano. No entanto, houve uma redução de células com baixo danos na comparação do D35 com o diagnóstico. Este resultado se confirmou em pacientes que apresentaram doença residual mínima positiva. A via de NER permaneceu constante, no entanto, em um único paciente, foi observada uma diminuição significativa da expressão dos genes, talvez devido ao silenciamento ou a regulação negativa das vias de reparo. Níveis de danos e reparação do DNA podem influenciar o resultado clínico, estar envolvidos na resistência aos fármacos e potencializar o risco de recidiva. Este é o primeiro estudo que avalia o dano ao DNA em amostras de MO de pacientes pediátricos com LLA. Apesar do pequeno número de pacientes alocados para o estudo, a partir dos achados é possível concluir que complexos de reparo merecem ser investigados a curto e a longo prazo. Acompanhamento dos resultados do paciente vai ajudar a elucidar a implicação dos nossos achados em taxas de cura e de recidiva. / Cancer is the leading cause of death by disease after 1 year old until the end of adolescence, except those related to accidents and violence. Acute Lymphoid Leukemia (ALL) affects lymphoid cells and worsens quickly. They are the most frequent tumors in childhood and account for a third of all malignancies in this age group. On average, the cure rate exceeds 70%, however, despite the progress of recent decades, rates of children with disease recurrence remains significant. Endogenous DNA damage occurs at a very high frequency, in addition to the damage caused by anti-tumor therapies. Change in the repair of DNA damage can induce resistance mechanisms to chemotherapy, resulting in increased repair of DNA lesions. Nucleotide Excision Repair (NER) pathway is the more versatile and flexible DNA repair in cells. Its components are being studied as prognostic biomarkers and targeted therapies. However, there are some reports of DNA damage in pediatric Acute Lymphoid Leukemia (ALL). In this study, we conducted an observational follow-up study in pediatric patients to assess DNA damage by alkaline comet assay and gene expression of NER pathway during induction chemotherapy. Bone marrow (BM) samples at diagnosis, 15th (D15) and 30th (D30) of treatment were collected from 28 patients with ALL. There was no increase in damage index. However, there was a reduction of cells with low damage in comparison to the D35 diagnosis. This result was confirmed in patients with positive minimal residual disease. The NER pathway remained constant, however, in one patient, a significant decrease of gene expression was observed perhaps due to the silencing or down-regulation of repair pathways. Damage levels and DNA repair can influence the clinical result and may be involved in drug resistance and enhance the risk of recurrence. This is the first study to assess DNA damage in BM samples of pediatric patients with ALL. Despite the small number of patients allocated to the study, from the findings we conclude that repair complex deserves to be investigated in the short and long term. Monitoring patient’s outcomes will help to access the implication of our findings in cure and relapse rates.

O câncer é a primeira causa de mortes por doença, após 1 ano de idade, até o final da adolescência, excetuando aquelas relacionadas aos acidentes e à violência. A Leucemia Linfoide Aguda (LLA) afeta células linfoides e agrava-se rapidamente. São os tumores mais frequentes na infância e representam um terço de todas as neoplasias malignas nesta faixa etária. Em média, a taxa de cura excede 70%, todavia, apesar dos avanços das últimas décadas, os índices de crianças que apresentam recidiva da doença continua significativo. Danos endógenos ao DNA ocorrem numa frequência altíssima, além dos danos causados por terapias antitumorais. Alteração no reparo ao dano do DNA pode induzir mecanismos de resistência ao tratamento quimioterápico, resultando em aumento do reparo de lesões do DNA. Reparo por Excisão de Nucleotídeos (NER) é a via de reparo de DNA mais versátil e flexível nas células. Seus componentes estão sendo estudados como biomarcadores de prognóstico e terapias-alvo. No entanto, alguns relatórios têm abordado danos de DNA em Leucemia Linfoide Aguda (LLA) pediátrica. Neste estudo, realizamos um estudo de acompanhamento observacional em pacientes pediátricos para avaliar os danos do DNA pelo Ensaio Cometa Alcalino e expressão gênica da via de NER durante a indução da quimioterapia. Amostras de medula óssea (MO) ao diagnóstico, dia 15 (D15) e 30 (D30) do tratamento foram coletadas de 28 pacientes com LLA. Não houve aumento no índice de dano. No entanto, houve uma redução de células com baixo danos na comparação do D35 com o diagnóstico. Este resultado se confirmou em pacientes que apresentaram doença residual mínima positiva. A via de NER permaneceu constante, no entanto, em um único paciente, foi observada uma diminuição significativa da expressão dos genes, talvez devido ao silenciamento ou a regulação negativa das vias de reparo. Níveis de danos e reparação do DNA podem influenciar o resultado clínico, estar envolvidos na resistência aos fármacos e potencializar o risco de recidiva. Este é o primeiro estudo que avalia o dano ao DNA em amostras de MO de pacientes pediátricos com LLA. Apesar do pequeno número de pacientes alocados para o estudo, a partir dos achados é possível concluir que complexos de reparo merecem ser investigados a curto e a longo prazo. Acompanhamento dos resultados do paciente vai ajudar a elucidar a implicação dos nossos achados em taxas de cura e de recidiva. / Cancer is the leading cause of death by disease after 1 year old until the end of adolescence, except those related to accidents and violence. Acute Lymphoid Leukemia (ALL) affects lymphoid cells and worsens quickly. They are the most frequent tumors in childhood and account for a third of all malignancies in this age group. On average, the cure rate exceeds 70%, however, despite the progress of recent decades, rates of children with disease recurrence remains significant. Endogenous DNA damage occurs at a very high frequency, in addition to the damage caused by anti-tumor therapies. Change in the repair of DNA damage can induce resistance mechanisms to chemotherapy, resulting in increased repair of DNA lesions. Nucleotide Excision Repair (NER) pathway is the more versatile and flexible DNA repair in cells. Its components are being studied as prognostic biomarkers and targeted therapies. However, there are some reports of DNA damage in pediatric Acute Lymphoid Leukemia (ALL). In this study, we conducted an observational follow-up study in pediatric patients to assess DNA damage by alkaline comet assay and gene expression of NER pathway during induction chemotherapy. Bone marrow (BM) samples at diagnosis, 15th (D15) and 30th (D30) of treatment were collected from 28 patients with ALL. There was no increase in damage index. However, there was a reduction of cells with low damage in comparison to the D35 diagnosis. This result was confirmed in patients with positive minimal residual disease. The NER pathway remained constant, however, in one patient, a significant decrease of gene expression was observed perhaps due to the silencing or down-regulation of repair pathways. Damage levels and DNA repair can influence the clinical result and may be involved in drug resistance and enhance the risk of recurrence. This is the first study to assess DNA damage in BM samples of pediatric patients with ALL. Despite the small number of patients allocated to the study, from the findings we conclude that repair complex deserves to be investigated in the short and long term. Monitoring patient’s outcomes will help to access the implication of our findings in cure and relapse rates.

O câncer é a primeira causa de mortes por doença, após 1 ano de idade, até o final da adolescência, excetuando aquelas relacionadas aos acidentes e à violência. A Leucemia Linfoide Aguda (LLA) afeta células linfoides e agrava-se rapidamente. São os tumores mais frequentes na infância e representam um terço de todas as neoplasias malignas nesta faixa etária. Em média, a taxa de cura excede 70%, todavia, apesar dos avanços das últimas décadas, os índices de crianças que apresentam recidiva da doença continua significativo. Danos endógenos ao DNA ocorrem numa frequência altíssima, além dos danos causados por terapias antitumorais. Alteração no reparo ao dano do DNA pode induzir mecanismos de resistência ao tratamento quimioterápico, resultando em aumento do reparo de lesões do DNA. Reparo por Excisão de Nucleotídeos (NER) é a via de reparo de DNA mais versátil e flexível nas células. Seus componentes estão sendo estudados como biomarcadores de prognóstico e terapias-alvo. No entanto, alguns relatórios têm abordado danos de DNA em Leucemia Linfoide Aguda (LLA) pediátrica. Neste estudo, realizamos um estudo de acompanhamento observacional em pacientes pediátricos para avaliar os danos do DNA pelo Ensaio Cometa Alcalino e expressão gênica da via de NER durante a indução da quimioterapia. Amostras de medula óssea (MO) ao diagnóstico, dia 15 (D15) e 30 (D30) do tratamento foram coletadas de 28 pacientes com LLA. Não houve aumento no índice de dano. No entanto, houve uma redução de células com baixo danos na comparação do D35 com o diagnóstico. Este resultado se confirmou em pacientes que apresentaram doença residual mínima positiva. A via de NER permaneceu constante, no entanto, em um único paciente, foi observada uma diminuição significativa da expressão dos genes, talvez devido ao silenciamento ou a regulação negativa das vias de reparo. Níveis de danos e reparação do DNA podem influenciar o resultado clínico, estar envolvidos na resistência aos fármacos e potencializar o risco de recidiva. Este é o primeiro estudo que avalia o dano ao DNA em amostras de MO de pacientes pediátricos com LLA. Apesar do pequeno número de pacientes alocados para o estudo, a partir dos achados é possível concluir que complexos de reparo merecem ser investigados a curto e a longo prazo. Acompanhamento dos resultados do paciente vai ajudar a elucidar a implicação dos nossos achados em taxas de cura e de recidiva. / Cancer is the leading cause of death by disease after 1 year old until the end of adolescence, except those related to accidents and violence. Acute Lymphoid Leukemia (ALL) affects lymphoid cells and worsens quickly. They are the most frequent tumors in childhood and account for a third of all malignancies in this age group. On average, the cure rate exceeds 70%, however, despite the progress of recent decades, rates of children with disease recurrence remains significant. Endogenous DNA damage occurs at a very high frequency, in addition to the damage caused by anti-tumor therapies. Change in the repair of DNA damage can induce resistance mechanisms to chemotherapy, resulting in increased repair of DNA lesions. Nucleotide Excision Repair (NER) pathway is the more versatile and flexible DNA repair in cells. Its components are being studied as prognostic biomarkers and targeted therapies. However, there are some reports of DNA damage in pediatric Acute Lymphoid Leukemia (ALL). In this study, we conducted an observational follow-up study in pediatric patients to assess DNA damage by alkaline comet assay and gene expression of NER pathway during induction chemotherapy. Bone marrow (BM) samples at diagnosis, 15th (D15) and 30th (D30) of treatment were collected from 28 patients with ALL. There was no increase in damage index. However, there was a reduction of cells with low damage in comparison to the D35 diagnosis. This result was confirmed in patients with positive minimal residual disease. The NER pathway remained constant, however, in one patient, a significant decrease of gene expression was observed perhaps due to the silencing or down-regulation of repair pathways. Damage levels and DNA repair can influence the clinical result and may be involved in drug resistance and enhance the risk of recurrence. This is the first study to assess DNA damage in BM samples of pediatric patients with ALL. Despite the small number of patients allocated to the study, from the findings we conclude that repair complex deserves to be investigated in the short and long term. Monitoring patient’s outcomes will help to access the implication of our findings in cure and relapse rates.

The sensitivity of Xeroderma pigmentosa (XP) patients to sunlight has spurred the discovery and genetic and biochemical analysis of the eight XP gene products (XPA-XPG plus XPV) responsible for this disorder. These studies also have served to elucidate the nucleotide excision repair (NER) process, especially the critical role played by the XPA protein. More recent studies have shown that NER also involves numerous other proteins normally employed in DNA metabolism and cell cycle regulation. Central among these is ataxia telangiectasia and Rad3-related (ATR), a protein kinase involved in intracellular signaling in response to DNA damage, especially DNA damage-induced replicative stresses. This review summarizes recent findings on the interplay between ATR as a DNA damage signaling kinase and as a novel ligand for intrinsic cell death proteins to delay damage-induced apoptosis, and on ATR’s regulation of XPA and the NER process for repair of UV-induced DNA adducts. ATR’s regulatory role in the cytosolic-to-nuclear translocation of XPA will be discussed. In addition, recent findings elucidating a non-NER role for XPA in DNA metabolism and genome stabilization at ds-ssDNA junctions, as exemplified in prematurely aging progeroid cells, also will be reviewed.