Characterization of the AP endonuclease enzyme APN-1 from C. elegans

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Stimulation of human 8-oxoguanine-DNA glycosylase by AP-endonuclease: potential coordination of the initial steps in base excision repair

8-Oxoguanine-DNA glycosylase 1 (OGG1), with intrinsic AP lyase activity, is the major enzyme for repairing 7,8-dihydro-8-oxoguanine (8-oxoG), a critical mutagenic DNA lesion induced by reactive oxygen species. Human OGG1 excised the damaged base from an 8-oxoG·C-containing duplex oligo with a very low apparent kcat of 0.1 min–1 at 37°C and cleaved abasic (AP) sites at half the rate, thus leaving abasic sites as the major product. Excision of 8-oxoG by OGG1 alone did not follow Michaelis–Menten kinetics. However, in the presence of a comparable amount of human AP endonuclease (APE1) the specific activity of OGG1 was increased ∼5-fold and Michaelis–Menten kinetics were observed. Inactive APE1, at a higher molar ratio, and a bacterial APE (Nfo) similarly enhanced OGG1 activity. The affinity of OGG1 for its product AP·C pair (Kd ∼ 2.8 nM) was substantially higher than for its substrate 8-oxoG·C pair (Kd ∼ 23.4 nM) and the affinity for its final β-elimination product was much lower (Kd ∼ 233 nM). These data, as well as single burst kinetics studies, indicate that the enzyme remains tightly bound to its AP product following base excision and that APE1 prevents its reassociation with its product, thus enhancing OGG1 turnover. These results suggest coordinated functions of OGG1 and APE1, and possibly other enzymes, in the DNA base excision repair pathway.

Characterization of uracil-DNA glycosylase activity from Trypanosoma cruzi and its stimulation by AP endonuclease

The intracellular pathogen Trypanosoma cruzi is the etiological agent of Chagas’ disease. We have isolated a full-length cDNA encoding uracil-DNA glycosylase (UDGase), a key enzyme involved in DNA repair, from this organism. The deduced protein sequence is highly conserved at the C-terminus of the molecule and shares key residues involved in binding or catalysis with most of the UDGases described so far, while the N-terminal part is highly variable. The gene is single copy and is located on a chromosome of ∼1.9 Mb. A His-tagged recombinant protein was overexpressed, purified and used to raise polyclonal antibodies. Western blot analysis revealed the existence of a single UDGase species in parasite extracts. Using a specific ethidium bromide fluorescence assay, recombinant T.cruzi UDGase was shown to specifically excise uracil from DNA. The addition of both Leishmania major AP endonuclease and exonuclease III, the major AP endonuclease from Escherichia coli, produces stimulation of UDGase activity. This activation is specific for AP endonuclease and suggests functional communication between the two enzymes.

Fractionated Radiation Exposure of Rat Spinal Cords Leads to Latent Neuro- Inflammation in Brain, Cognitive Deficits,and Alterations in Apurinic Endonuclease 1

Ionizing radiation causes degeneration of myelin, the insulating sheaths of neuronal axons, leading to neurological impairment. As radiation research on the central nervous system has predominantly focused on neurons, with few studies addressing the role of glial cells, we have focused our present research on identifying the latent effects of single/ fractionated -low dose of low/ high energy radiation on the role of base excision repair protein Apurinic Endonuclease-1, in the rat spinal cords oligodendrocyte progenitor cells’ differentiation. Apurinic endonuclease-1 is predominantly upregulated in response to oxidative stress by low- energy radiation, and previous studies show significant induction of Apurinic Endonuclease-1 in neurons and astrocytes. Our studies show for the first time, that fractionation of protons cause latent damage to spinal cord architecture while fractionation of HZE (28Si) induce increase in APE1 with single dose, which then decreased with fractionation. The oligodendrocyte progenitor cells differentiation was skewed with increase in immature oligodendrocytes and astrocytes, which likely cause the observed decrease in white matter, increased neuro-inflammation, together leading to the observed significant cognitive defects.

Análise filogenética e funcional de dois genes de reparo homólogos a AP endonuclease em cana-de-açúcar: ScARP1 e ScARP3

The genome of all organisms constantly suffers the influence of mutagenic factors from endogenous and/or exogenous origin, which may result in damage for the genome. In order to keep the genome integrity there are different DNA repair pathway to detect and correct these lesions. In relation to the plants as being sessile organisms, they are exposed to this damage frequently. The Base Excision DNA Repair (BER) is responsible to detect and repair oxidative lesions. Previous work in sugarcane identified two sequences that were homologous to Arabidopsis thaliana: ScARP1 ScARP3. These two sequences were homologous to AP endonuclease from BER pathway. Then, the aim of this work was to characterize these two sequence using different approaches: phylogenetic analysis, in silico protein organelle localization and by Nicotiana tabacum transgenic plants with overexpression cassette. The in silico data obtained showed a duplication of this sequence in sugarcane and Poaceae probably by a WGD event. Furthermore, in silico analysis showed a new localization in nuclei for ScARP1 protein. The data obtained with transgenic plants showed a change in development and morphology. Transgenic plants had slow development when compared to plants not transformed. Then, these results allowed us to understand better the potential role of this sequence in sugarcane and in plants in general. More work is important to be done in order to confirm the protein localization and protein characterization for ScARP1 and ScARP3

Caracterização de dois cDNAs homológos e uma AP endonuclease em cana-de-açúcar

The genome of all organisms is subject to injuries that can be caused by endogenous and environmental factors. If these lesions are not corrected, it can be fixed generating a mutation which can be lethal to the organisms. In order to prevent this, there are different DNA repair mechanisms. These mechanisms are well known in bacteria, yeast, human, but not in plants. Two plant models Oriza sativa and Arabidopsis thaliana had the genome sequenced and due to this some DNA repair genes have been characterized. The aim of this work is to characterized two sugarcane cDNAs that had homology to AP endonuclease: scARP1 and scARP3. In silico has been done with these two sequences and other from plants. It has been observed domain conservation on these sequences, but the cystein at 65 position that is a characteristic from the redox domain in APE1 protein was not so conservated in plants. Phylogenetic relationship showed two branches, one branch with dicots and monocots sequence and the other branch with only monocots sequences. Another approach in order to characterized these two cDNAs was to construct overexpression cassettes (sense and antisense orientation) using the 35S promoter. After that, these cassettes were transferred to the binary vector pPZP211. Furthermore, previously in the laboratory was obtained a plant from nicotiana tabacum containing the overexpression cassette in anti-sense orientation. It has been observed that this plant had a slow development and problems in setting seeds. After some manual crossing, some seeds were obtained (T2) and it was analyzed the T2 segregation. The third approach used in this work was to clone the promoter region from these two cDNAs by PCR walking. The sequences obtained were analyzed using the program PLANTCARE. It was observed in these sequences some motives that may be related to oxidative stress response

Structural basis of nuclear import of flap endonuclease 1 (FEN1)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Untersuchungen zur Beeinflussung der Reparatur oxidativer DNA-Schäden durch Poly(ADP-Ribose)-Polymerase, AP-Endonuklease 1 und das Xeroderma pigmentosum A Protein

Gegenstand dieser Arbeit war die Untersuchung der Bedeutung der Poly(ADP-Ribose)-Polymerase 1 (PARP 1), der AP Endonuklease 1 (Ape 1) und des Xeroderma pigmentosum A (XPA) Proteins für die DNA-Reparatur in Säugerzellen.Zunächst wurde der Einfluss der PARP 1-Aktivität auf die Reparatur verschiedener DNA-Modifikationen untersucht. Die Ergebnisse zeigen erstmalig, dass eine Hemmung der PARP-Aktivität nicht nur eine deutliche Verlangsamung der Reparatur von Einzelstrangbrüchen, sondern auch von oxidativen Purinmodifikationen und Pyrimidindimeren zur Folge hat. Interessanterweise erfolgte diese Verlangsamung der DNA-Reparatur nicht in Csb-defizienten Zellen. Diese Ergebnisse deuten darauf hin, dass die Aktivierung der PARP 1 und das Csb-Protein zusammen an einem neuartigen Mechanismus beteiligt sind, der die globale Reparatur verschiedener DNA-Modifikationen beschleunigt.Weiterhin wurde die Bedeutung der Nukleotidexcisionsreparatur als back-up Reparatur von 8 Hydroxyguanin untersucht. Dazu wurden normale und XPA-defiziente Fibroblasten des Menschen mit einem hOgg1-anitsense Konstrukt transfiziert und dann in diesen Zellen die Reparaturkinetiken oxidativer Basenmodifikationen bestimmt. Dadurch konnte eine Beteiligung des XPA-Proteins an diesem Reparaturweg ausgeschlossen werden.Außerdem wurden die Auswirkungen einer AP Endonuklease-1-Überexpression in XRCC1-defizienten Zellen auf die Reparatur von Einzelstrangbrüchen untersucht. Die Reparatur der induzierten Einzelstrangbrüche war in XRCC1-defizienten Zellen erwartungsgemäß deutlich langsamer als in XRCC1-profizienten Zellen. Die Überexpression der AP Endonuklease 1 in XRCC1-defizienten Zellen führte zu einer teilweisen Beschleunigung der Einzelstrangbruchreparatur.

CULTO IMPERIAL E O APOCALIPSE DE JOÃO Uma análise exegética de Ap 13,1-18

Esta dissertação investiga a narrativa de visão apocalíptica encontrada em Ap 13,1-18. Ela parte da pergunta sobre a realidade que influenciou o autor no momento da composição, utilizando-se, assim, de uma linguagem provocativa. Nossa hipótese indica que o Apocalipse de João, importante fonte das experiências do Cristianismo primitivo no final do primeiro século, proporciona uma dura crítica às exigências de adoração dirigidas às autoridades romanas através do Culto Imperial. As imagens das bestas descritas em Ap 13,1-18 expressam este tema através de uma linguagem provocativa, fundamentada na força da palavra e na tradição do mito do antagonista encontrado no Antigo Oriente Próximo. Neste sentido, acreditamos que o autor demoniza e estigmatiza as expressões da religião oficial, as quais tratam os governantes como seres divinos, e os promotores do Culto Imperial na região da Ásia Menor.(AU)

CULTO IMPERIAL E O APOCALIPSE DE JOÃO Uma análise exegética de Ap 13,1-18

Esta dissertação investiga a narrativa de visão apocalíptica encontrada em Ap 13,1-18. Ela parte da pergunta sobre a realidade que influenciou o autor no momento da composição, utilizando-se, assim, de uma linguagem provocativa. Nossa hipótese indica que o Apocalipse de João, importante fonte das experiências do Cristianismo primitivo no final do primeiro século, proporciona uma dura crítica às exigências de adoração dirigidas às autoridades romanas através do Culto Imperial. As imagens das bestas descritas em Ap 13,1-18 expressam este tema através de uma linguagem provocativa, fundamentada na força da palavra e na tradição do mito do antagonista encontrado no Antigo Oriente Próximo. Neste sentido, acreditamos que o autor demoniza e estigmatiza as expressões da religião oficial, as quais tratam os governantes como seres divinos, e os promotores do Culto Imperial na região da Ásia Menor.(AU)

Mechanism of stimulation of the DNA glycosylase activity of hOGG1 by the major human AP endonuclease: bypass of the AP lyase activity step

The generation of reactive oxygen species in the cell provokes, among other lesions, the formation of 8-oxo-7,8-dihydroguanine (8-oxoG) in DNA. Due to mispairing with adenine during replication, 8-oxoG is highly mutagenic. To minimise the mutagenic potential of this oxidised purine, human cells have a specific 8-oxoG DNA glycosylase/AP lyase (hOGG1) that initiates the base excision repair (BER) of 8-oxoG. We show here that in vitro this first enzyme of the BER pathway is relatively inefficient because of a high affinity for the product of the reaction it catalyses (half-life of the complex is >2 h), leading to a lack of hOGG1 turnover. However, the glycosylase activity of hOGG1 is stimulated by the major human AP endonuclease, HAP1 (APE1), the enzyme that performs the subsequent step in BER, as well as by a catalytically inactive mutant (HAP1-D210N). In the presence of HAP1, the AP sites generated by the hOGG1 DNA glycosylase can be occupied by the endonuclease, avoiding the re-association of hOGG1. Moreover, the glycosylase has a higher affinity for a non-cleaved AP site than for the cleaved DNA product generated by HAP1. This would shift the equilibrium towards the free glycosylase, making it available to initiate new catalytic cycles. In contrast, HAP1 does not affect the AP lyase activity of hOGG1. This stimulation of only the hOGG1 glycosylase reaction accentuates the uncoupling of its glycosylase and AP lyase activities. These data indicate that, in the presence of HAP1, the BER of 8-oxoG residues can be highly efficient by bypassing the AP lyase activity of hOGG1 and thus excluding a potentially rate limiting step.

CULTO IMPERIAL E O APOCALIPSE DE JOÃO Uma análise exegética de Ap 13,1-18

Esta dissertação investiga a narrativa de visão apocalíptica encontrada em Ap 13,1-18. Ela parte da pergunta sobre a realidade que influenciou o autor no momento da composição, utilizando-se, assim, de uma linguagem provocativa. Nossa hipótese indica que o Apocalipse de João, importante fonte das experiências do Cristianismo primitivo no final do primeiro século, proporciona uma dura crítica às exigências de adoração dirigidas às autoridades romanas através do Culto Imperial. As imagens das bestas descritas em Ap 13,1-18 expressam este tema através de uma linguagem provocativa, fundamentada na força da palavra e na tradição do mito do antagonista encontrado no Antigo Oriente Próximo. Neste sentido, acreditamos que o autor demoniza e estigmatiza as expressões da religião oficial, as quais tratam os governantes como seres divinos, e os promotores do Culto Imperial na região da Ásia Menor.(AU)

Análise filogenética e funcional de dois genes de reparo homólogos a AP endonuclease em cana-de-açúcar: ScARP1 e ScARP3

The genome of all organisms constantly suffers the influence of mutagenic factors from endogenous and/or exogenous origin, which may result in damage for the genome. In order to keep the genome integrity there are different DNA repair pathway to detect and correct these lesions. In relation to the plants as being sessile organisms, they are exposed to this damage frequently. The Base Excision DNA Repair (BER) is responsible to detect and repair oxidative lesions. Previous work in sugarcane identified two sequences that were homologous to Arabidopsis thaliana: ScARP1 ScARP3. These two sequences were homologous to AP endonuclease from BER pathway. Then, the aim of this work was to characterize these two sequence using different approaches: phylogenetic analysis, in silico protein organelle localization and by Nicotiana tabacum transgenic plants with overexpression cassette. The in silico data obtained showed a duplication of this sequence in sugarcane and Poaceae probably by a WGD event. Furthermore, in silico analysis showed a new localization in nuclei for ScARP1 protein. The data obtained with transgenic plants showed a change in development and morphology. Transgenic plants had slow development when compared to plants not transformed. Then, these results allowed us to understand better the potential role of this sequence in sugarcane and in plants in general. More work is important to be done in order to confirm the protein localization and protein characterization for ScARP1 and ScARP3

Functional complementation of Leishmania (Leishmania) amazonensis AP endonuclease gene (lamap) in Escherichia coli mutant strains challenged with DNA damage agents

During its life cycle Leishmania spp. face several stress conditions that can cause DNA damages. Base Excision Repair plays an important role in DNA maintenance and it is one of the most conserved mechanisms in all living organisms. DNA repair in trypanosomatids has been reported only for Old World Leishmania species. Here the AP endonuclease from Leishmania (L.) amazonensis was cloned, expressed in Escherichia coli mutants defective on the DNA repair machinery, that were submitted to different stress conditions, showing ability to survive in comparison to the triple null mutant parental strain BW535. Phylogenetic and multiple sequence analyses also confirmed that LAMAP belongs to the AP endonuclease class of proteins.