Compartimentación de la replicación del DNA el bacteriófago O29: función de la proteína p1
Author
Serrano de las Heras, GemmaAdvisor
Bravo García, AliciaEntity
UAM. Departamento de Biología MolecularDate
2004-12-10Subjects
Bacteriófagos - Tesis doctorales; ADN - Replicación - Tesis doctorales; Biología y Biomedicina / BiologíaNote
Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular, Fecha de lectura 10-12-2004Abstract
In eukaryotes, the use of large organizing structures to bring together replication factors seems to be a general mechanism to enhance the efficiency of the replication process. However, the mechanisms underlying , compartmentalization of prokaryotic DNA replication are largely unknown. In the case of Bacillus subtilis phage (P29, the viral protein pl (85 residues) enhances the rate of in vivo (P29 DNA replication, playing a critica1 role when bacteria are growing at high, rather than low, temperatures. Previous cell fractionation studies indicated that pl is membrane-associated both during
synthesis of Q29 DNA and in the absence of other viral components. We have
studied the nature of this association by phase partitioning with Triton X-114,
a non ionic detergent widely used for the isolation of integral membrane proteins. This biochemical analysis showed that pl, like integral membrane proteins, has an amphiphilic nature. Furthermore, immunoelectron microscopy studies revealed that protein pl is located at or close to the bacterial membrane. By combining in vivo chemical cross-linking and cell fractionation techniques, we demonstrated that pl assembies into membraneassociated multimeric structures during viral DNA replication. These structures are also formed when initiation of Q29 DNA replication is blocked due to the lack of viral replisome components. In addition, protein pl encoded by plasmid generates membrane-associated multimers and supports DNA replication of a pl-lacking mutant phage, suggesting that the preassembled structures are functional. These results support an in vivo $129 DNA replication model in which a pl multimeric structure assembled on
the bacterial membrane provides an anchoring cite for the $29 replication complexes.
Previous studies demonstrated that protein pl also self-interacts in vitro, generating highly ordered structures. Specifically, protein plAN33, which retains the sequence of pl panning residues Met34 to Lys85, assembles into two-dimensional protofilament sheets. The region of protein pl located between amino acids Glu38 and Asn65 presumably forms an a-helical coiledcoi1 structure. We have examined the role of this coiled-coi1 sequence in the formation of protofilament sheets. Using sedimentation assays and negative stain electron microscopy analysis we demonstrated that residues Leu46, Met53 and Leu60, but not Leu39, are essential for plAN33 assembly into sheets. Remarkably, replacement of Leu46 by Val shifts the pathway of molecular assembly, leading to the formation of filamentous polymers -10 nm in diameter. These results show that a short coiled-coi1 motif (-3 heptad repeats) functions to assemble a small protein into two-dimensional sheets.
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