Dinámica de la topología del DNA durante la replicación

Abstract

Comprender cómo replica el DNA es una cuestión crítica para entender la vida.La replicación del DNA es un proceso muy complejo y altamente regulado, en el queestán implicadas una gran cantidad de moléculas. Hoy por hoy, este proceso no estáplenamente caracterizado. En la presente Tesis Doctoral se ha intentado profundizar enla comprensión de la replicación del DNA utilizando diferentes aproximaciones a lo largode sus distintas etapas.Actualmente no existe un consenso entre los miembros de la comunidad científicaacerca de si las horquillas replicativas pueden girar libremente in vivo durante lareplicación con la consecuente formación de pre-encadenados. En una primera fase de lapresente Tesis Doctoral se aportó información acerca de esta cuestión, tratando decontestar a la pregunta de si las horquillas pueden girar durante la replicación. Para ellose analizó la topología de tres plásmidos bacterianos derivados de pBR322 que conteníanla secuencia para la terminación TerE clonada a distintas distancias del origen dereplicación. Con estos plásmidos se transformaron dos estirpes distintas de E. coli, unaproficiente y otra deficiente en la Topoisomerasa IV (Topo IV). Se aislaronintermediarios de replicación (RIs) que se analizaron mediante electroforesisbidimensional en geles de agarosa. Los resultados obtenidos indican que los RIs aisladosde las células con la Topo IV inactiva presentan un nivel de torsión mayor que los aisladosde la estirpe con la Topo IV activa. Esta observación sugiere que las horquillas puedengirar libremente in vivo a medida que avanza la replicación...

How DNA replicates is a critical question to understand life. The replicationprocess is very complex and is highly regulated. Many molecules are implicated in thisprocess which is not fully characterized. In this Doctoral Thesis, we tried to improve theknowledge about replication using different approaches.Nowadays, in the scientific community, there is not a consensus as to wether ornot swiveling of the replication fork and formation of pre-catenanes can occur in vivo. Inthe first part of this Thesis we tried to find out if replication forks can swivel duringreplication. To achieve this goal, we examined the topology of three bacterial plasmidswith the fork stalled at different sites after initiation. These plasmids were used totransform two different E. coli strain, one of which had Topoisomerase IV (Topo IV)active in vivo and the other one had it inactive. We isolated replication intermediates (RIs)which were analyzed by high-resolution two-dimensional agarose gel electrophoresis.The results obtained indicated that RIs were more torsionally tensioned when they wereisolated from cells without Topo IV. This observation strongly suggests that forks canswivel in vivo leading to the formation of pre-catenanes as replication progresses.In a second part, we studied different characteristics about replication fork barriers(RFBs). These barriers are in the rDNA of all eukaryotic organisms and they are veryimportant for the cells because in the rDNA transcription and replication occur at thesame time. If there would be no RFBs, the replication and transcription machineries couldcollide. RFBs block those replication forks moving in the opposite direction totranscription. We investigated and quantified the efficiency of RFBs to stall replicationforks. To this end, we transfected S. cerevisiae cells with circular artificial yeastminichromosomes containing different copies of RFBs. To identify fork stalling we usedtwo-dimensional agarose gel electrophoresis to analyze the RIs. We obtained severalresults that allowed us to conclude that blockage of replication forks at the buddingyeast’s RFBs when occurred were permanent, but this blockage only occurred in somecases...