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dc.contributor.advisorHermoso, José Miguel-
dc.contributor.authorAlcorlo, Martín-
dc.descriptionTesis Doctoral inédita leida en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura 30-03-2007en_US
dc.description.abstractProtein p6 of Bacillus subtilis bacteriophage Φ29 is essential for phage development. It activates the initiation of DNA replication and promotes the early to late transcriptional switch. These activities require the formation of a nucleoprotein complex in which the DNA forms a right-handed superhelix wrapping around a multimeric protein core. However, there was no evidence of p6 binding to Φ29 DNA in vivo. In this work, protein p6 binding to DNA has been studied by crosslinking, chromatin immunoprecipitation (X-ChIP) and real-time PCR. The results have shown that protein p6 binds to most, if not all, the viral genome in vivo, although with higher affinity for both DNA ends, which contain the replication origins. The p6 binding to different Φ29 DNA regions is modulated by the structural properties of their nucleotide sequences and the higher affinity for DNA ends is possibly related to the presence of sequences in which their bendability properties favour the formation of the p6-DNA complex. Moreover, protein p6 binds to Φ29 DNA with higher affinity than plasmid DNA. This specificity could be due to the p6 binding preference for less negatively supercoiled DNA. Thus, the p6 binding dependence on DNA topology could explain its preferential binding to viral with respect to plasmid or bacterial DNA, whose level of negative supercoiling is presumably higher than that of Φ29 DNA. Protein p6 binding to Φ29 DNA is greatly increased when negative supercoiling is decreased by novobiocin. The fact that gyrase is required for viral DNA replication indicates that, although non-covalently closed, the viral genome is topologically constrained in vivo. Viral proteins p1 and p17 seem to be required to restrain the proper topology of Φ29 DNA. Binding of protein p6 all along the Φ29 genome strongly suggests that its functions in replication and transcription control could be outcomes of a more global role as a histone-like protein. The in vivo interaction with DNA of protein p6 of the Φ29-related bacteriophage GA-1 has been also studied with the finding that it has a much lower dependence on supercoiling than Φ29 p6 and that is able to bind to Φ29 DNA, although it does not functionally replace the Φ29 counterpart. We have taken advantage of the binding properties of protein p6 to monitor the entry of Φ29 DNA into the cell by X-ChIP, measuring p6 binding to both viral genome ends. With this approach, we have studied the Φ29 DNA ejection mechanism, demonstrating that the early viral membrane protein p16.7 is required, together with protein p17, for efficient execution of the second pull step of Φ29 DNA ejection. Finally, the quantitative in vitro characterization of reversible protein p6- DNA association in solution, in terms of stoichiometry, affinity and cooperativity, has been carried out using analytical ultracentrifugation. This analysis has shown that p6-DNA complex formation could be fitted by the empirical Hill function. The effect of macromolecular crowding on the p6-DNA complex formation has been also studied using sedimentation equilibrium analysis.en_US
dc.description.sponsorshipEl presente trabajo se ha llevado a cabo en el Centro de Biología Molecular “Severo Ochoa” bajo la dirección del Doctor José Miguel Hermoso Núñez y la tutoría de la Doctora Margarita Salas Falgueras. Para su realización se ha contado con una beca de formación de profesorado universitario del Ministerio de Educación y Ciencia.en_US
dc.format.extent29780042 bytes-
dc.publisherUniversidad Autónoma de Madriden_US
dc.subjectBacteriófago phi 29en_US
dc.titleEstudio de la interacción de la proteína p6 con el DNA del bacteriófago [phi] 29 de "Bacillus subtilis"en_US
dc.typetesis doctoralen_US
dc.description.peerreviewedPeer revieweden_US
item.openairetypetesis doctoral-
item.fulltextWith Fulltext-
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