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Previous issue date: 2007-02-23 === Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico === Plants are organisms sessile and because of this they are susceptible to genotoxic effects due to environmental exposure such as light [including ultraviolet (UV)], heat, drought and chemicals agents. Therefore, there are differents pathways in order to detect a lesion and correct. These pathways are not well known in plants. The MutM/Fpg protein is a DNA glycosylase that is responsible for detect and correct oxidative lesions. In the sugarcane genome, it was found two possible cDNAs that had homology to this protein: scMUTM1 and scMUTM2. The aim of this work was to characterize the role of these cDNAs in plants. In order to do this, the expression level after oxidative stress was evaluated by semiquantitative RT-PCR. Another point analyzed in order to obtain the full-length gene, it was to use a sugarcane genomic library that was hybridized with both cDNAs as a probe. It was found two clones that will bought and sequenced. The promoter region was also cloned. It was obtained sequences only for scMUTM2 promoter region. The sequences obtained were divided into six groups. It was found regulatory motifs such as TATA-box, CAAT-box, oxidative stress element response and regulatory regions that response to light. The other point analyzed was to characterize the N-terminal region by PCR constructs. These constructs have deletions at 5 region. These sequences were introduce into Escherichia coli wild type strain (CC104) and double mutant (CC104mutMmutY). The results showed that proteins with deletions of scMUTM1 N-terminal region were able to complement the Fpg and MutY-glycosylase deficiency in CC104 mutMmutY reducing the spontaneous mutation frequency === As plantas s?o organismos particularmente suscept?veis aos efeitos genot?xicos devido a sua constante exposi??o a agentes do meio ambiente tais como a luz [incluindo a ultravioleta (UV)], o calor, a seca e diferentes subst?ncias qu?micas. Entretanto, existem diferentes mecanismos de reparo para detectar estas les?es e corrigi-las. Entretanto, em plantas estas vias s?o pouco compreendidas. Uma das vias ? a por excis?o de bases (BER). A prote?na MUTM/FPG ? uma DNA glicosilase da via BER que corrige danos oxidativos. No genoma da cana-de-a??car foram encontrados dois poss?veis cDNAs que possuem homologia de seq??ncia a esta prote?na: scMUTM1 e scMUTM2. O objetivo deste trabalho foi caracterizar o papel destes genes em plantas. Para isto, o n?vel de express?o dos genes ap?s estresse oxidativo foi avaliado atrav?s de RT-PCR semiquantitativo. Para obter a sequ?ncia completa dos gene, uma biblioteca de DNA gen?mico de cana-de-a??car foi hibridizada com sondas radioativas para ambos cDNAs, encontrando-se dois clones em potencial. A regi?o promotora foi clonada, seq?enciada e analisada. Dentro das seq??ncias obtidas para a regi?o promotora de scMUTM2, estas podem ser divididas em seis grupos. Os motivos regulat?rios foram identificados, tais como o TATA-box, CAAT-box, elementos de resposta ao estresse oxidativo e elementos de resposta ? luz. Um outro ponto de interesse neste trabalho foi a caracteriza??o da regi?o N-terminal da prote?na scMUTM1. Para isto, foram realizadas algumas constru??es via PCR. Estas constru??es foram posteriormente introduzidas em cepas selvagem (CC104) e duplo mutante (CC104mutMmutY) de Escherichia coli e foram realizados ensaios de complementa??o de modo a avaliar a import?ncia de cada regi?o para o reconhecimento e corre??o das les?es. Os resultados mostraram que as prote?nas com dele??es na por??o N-terminal de scMUTM1 s?o capazes de complementar a defici?ncia de Fpg and MutY-glicosilase na bact?ria CC104 mutMmutY reduzindo assim a freq??ncia de muta??o espont?nea
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