Caracterização Funcional e Determinação da Estrutura Tridimensional por Cristalografia de Raios X da Proteína RecA de Herbaspirillum seropedicae

• Ano de defesa: 2016
• Autor(a) principal: Leite, Wellington Claiton
• Orientador(a): Saab, Sérgio da Costa
• Banca de defesa: Brinatti, André Maurício , Benelli, Elaine , Chubatsu, Leda Satie , Andrade, André Vitor Chaves de
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: UNIVERSIDADE ESTADUAL DE PONTA GROSSA
• Programa de Pós-Graduação: Programa de Pós-Graduação em Ciências
• Departamento: Fisica
• País: BR
• Palavras-chave em Português: RecA; cristalografia; filamento de nucleoproteína RecA; atividade ATP básica; reação de troca de fitas; estrutura cristalina; análise estrutural
• Palavras-chave em Inglês: RecA; crystallography; RecA nucleoprotein flament; ATPase activity; DNA strand exchange; crystal structure; structural analysis
• Área do conhecimento CNPq: CNPQ::CIENCIAS EXATAS E DA TERRA::FISICA
• Link de acesso: http://tede2.uepg.br/jspui/handle/prefix/863

The bacterial RecA protein plays a role in the complex system of DNA damage repair. In the presence of ATP, RecA proteins polymerize onto single-strand DNA (ssDNA) as righthanded helical nucleoprotein laments, and catalyze strand exchange reaction between the ssDNA and homologous double-strand DNA (dsDNA) molecules. These activities are supported or stimulated by accessory proteins, as the single-stranded binding protein (SSB).Here, we report a functional and structural characterization of the Herbaspirillum seropedicae RecA protein (HsRecA).We report the crystal structure of HsRecA-ADP/ATP complex to 1.7 Å of atomic resolution. HsRecA protein contains a small N-terminal domain, a central core ATPase domain and a large C-terminal domain, similarly to homologous RecA proteins. Comparative structural analysis showed that the N-terminal polymerization motif of archaeal and eukaryotic RecA family proteins are also present in bacterial RecAs. The bacterial polymerization motif contains the sequence SV/IMR/KLG which interacts with the core ATPase domain residues DNLLLV/CS. In the inactive RecA, it is a loop - strand interaction, respectively, while in the active RecA it becomes a dyad strand. In both RecA forms, the polymerization motif seems to stabilize the subunitsubunit interface by hydrophobic interactions. The methionine of this motif may play an important role in the stability and formation of a right-handed helical nucleoprotein lament. The ATPase activity and the structure of the nucleoprotein lament of HsRecA and Escherichia coli RecA (EcRecA) were analyzed in the presence and absence of SSB. When SSB was added after RecA+ssDNA, HsRecA and EcRecA showed similar ATPase activity and nucleolament structure. However, when SSB was either not included or it was added before RecA+ssDNA, the HsRecA showed higher ATPase activity and formed longer nucleoprotein laments than EcRecA. Thus, HsRecA protein is more ecient at displacing SSB from ssDNA than EcRecA protein. HsRecA promoted DNA exchange more eficiently: a greater yield of nicked circular products were obtained in a shorter time. Reconstruction of electrostatic potential from the hexameric structure of HsRecAADP/ ATP revealed a high positive charge along the inner side, which is consistent with the fact that ssDNA binds inside the filament. It may explain the enhance capacity of HsRecA protein to bind ssDNA, forming a contiguous nucleoprotein filament, displace SSB and promote eficiently the DNA strand exchange reaction. Keywords: RecA, Crystallography, RecA nucleoprotein filament, ATPase activity, DNA strand exchange, crystal structure, structural analysis.