UNVEILING THE ROLE OF DPS, ENDOIII AND PPK FOR DNA PROTECTION AND REPAIR IN DEINOCOCCUS RADIODURANS UPON EXPOSURE TO GENOTOXIC STRESSure t

• Autor(a) principal: Martins, Guilherme David Castro
• Data de Publicação: 2022
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/154798
• topic: Deinococcus radiodurans; DNA repair; DNA protection; Oxidative stress; Ultraviolet radiation; Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias

Deinococcus radiodurans (Dr) is known for showing great resistance to ionizing radiation, and desiccation amongst other extreme conditions. This bacterium possesses different groups of proteins which are involved in the response to several stress agents by either protecting the DNA from suffering damage, repairing efficiently the damaged DNA or even being involved in the metabolism small antioxidant molecules, capable of protecting the proteome so that it can continue working under stress conditions. In this work we focused on two DNA binding protein under starved conditions (Dps1 and Dps2) present in Dr, which are able to bind and protect DNA against damage, on three Endonuclease III proteins (EndoIII-1, EndoIII-2 and EndoIII-3) which remove oxidation damaged bases in DNA through the Base Excision Repair (BER) pathway and on two Polyphosphate kinases (PPK1 and PPK2) that are responsible for the metabolism of inorganic polyphosphate (PolyP) which is involved in the defense against oxidative stress. To understand the role of these proteins and their possible interplay, we constructed and studied the resistance to stress of both single, double and triple knockout mutants, of each of these proteins, through the Tripartite Ligation Method, using Overlap PCR. The induced stress was exposure to UV-C radiation, hydrogen peroxide and methyl viologen. The stress response was compared with the wild type bacterium. Moreover, a single amino acid mutation of EndoIII-2 was generated in order to attempt its crystallization, to understand its structure and the molecular mechanisms involved in its activity.