RESUMO
Genotoxic stress results in more than 50 000 damaged DNA sites per cell per day. During DNA replication, processive high-fidelity DNA polymerases generally stall at DNA lesions and have to be displaced by translesion synthesis DNA polymerases, which are able to bypass the lesion. This switch is mediated by mono-ubiquitination of the processivity factor proliferating cell nuclear antigen (PCNA). To further investigate the regulation of the DNA polymerase exchange, we developed an easy and efficient method to synthesize site-specifically mono-ubiquitinated PCNA by click chemistry. By incorporating artificial amino acids that carry an azide (Aha) or an alkyne (Plk) in their side chains, into ubiquitin (Ub) and PCNA, respectively, we were able to link the two proteins site-specifically by the Cu(I) -catalyzed azide-alkyne cycloaddition. Finally, we show that the synthetic PCNA-Ub is able to stimulate DNA synthesis by DNA polymerase δ, and that DNA polymerase η has a higher affinity for PCNA-Ub than to PCNA.
Assuntos
DNA/química , Mimetismo Molecular , Antígeno Nuclear de Célula em Proliferação/química , Ubiquitina/química , Sequência de Aminoácidos , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Many proteins are post-translationally modified by the attachment of poly-ubiquitin (Ub) chains. Notably, the biological function of the attached Ub chain depends on the specific lysine residue used for conjugate formation. Here, we report an easy and efficient method to synthesize site-specifically linked Ub dimers by click reaction between two artificial amino acids. In fact, we were able to synthesize all seven naturally occurring Ub connectivities, providing the first example of a method that gives access to all Ub dimers. Furthermore, these synthetic Ub dimers are recognized by the natural ubiquitination machinery and are proteolytically stable, making them optimal candidates to further investigate the function of differently linked Ub chains.
Assuntos
Ubiquitina/síntese química , Sequência de Aminoácidos , Dimerização , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Ubiquitina/químicaRESUMO
Titanocene difluorides can be obtained by halide metathesis of the respective titanocene dichlorides with trimethyltin fluoride (Me(3)SnF), giving access to a new class of cytotoxic active substances. Furthermore, an improved method for the synthesis of diaryl-substituted titanocene dichlorides is presented.
Assuntos
Proliferação de Células/efeitos dos fármacos , Compostos Organometálicos/síntese química , Compostos Organometálicos/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Concentração Inibidora 50 , Neoplasias Renais/tratamento farmacológico , Células LLC-PK1 , Masculino , Camundongos , Camundongos Nus , Estrutura Molecular , Transplante de Neoplasias , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-bcl-2 , Relação Estrutura-Atividade , SuínosRESUMO
The conjugation of poly-ubiquitin chains is a widespread post-translational modification of proteins that plays a role in many different cellular processes. Notably, the biological function of the attached ubiquitin chain depends on which lysine residue is used for chain formation. Here, we report a method for the modular synthesis of site-specifically linked ubiquitin dimers, which is based on click reaction between two artificial amino acids. In this way, it is possible to synthesize all seven naturally occurring ubiquitin connectivities, thus giving access to all ubiquitin dimers. Furthermore, this method can be generally applied to link ubiquitin to any substrate protein or even to link any two proteins site specifically.