Atomic Force Microscopy characterization of DNA-binding proteins involved in the repair and organisation of DNA

Abstract

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Física de la Materia Condensada. Fecha de lectura: 28-11-2019Esta tesis tiene embargado el acceso al texto completo hasta el 28-05-2021The invention of the Atomic Force Microscope played a fundamental role in the characterization of nanometric molecular complexes. The Atomic Force Microscope is a suitable research technique for the study of biomolecules one by one with the advantage to be an effective technique inside and outside a liquid medium. However, its use in biology has been limited by the availability of faster visualization techniques which do not need to deposit a sample on top of a flat surface. In this thesis I present the result obtained in the study of three biological processes from a biophysical point of view with Atomic Force Microscopes. In the first project, three centromere-binding proteins are studied in the context of their plasmid partition systems. Plasmid partition systems consist of a group of proteins and DNA-sequences employed for the transmission and stabilization of plasmid molecules during cellular division. The three organisms, B. subtilis, C. crescentus and C. botulinum, have developed their partition systems differently. Regardless, these systems consist of a centromeric sequence near the origin of replication, a centromeric-binding protein and a NTPase motor protein. The formation of a segrosome complex, Centromeric-Binding protein bound to DNA, is showed by Atomic Force Microscopy images revealing a very different structure for the three biological organisms. In the second project, two protein of the Homologous Recombination mechanism are studied. The first, CtIP, is a tetrameric protein with some studies pointing to a short-resection of DNA-ends activity. The second, Dna2, is a helicase/nuclease protein with a defined role in the long-resection of DNA-ends in Homologous Recombination. However, the binding-mode of these proteins to DNA molecules is not completely understood. Here, I present the data obtained on CtIP and Dna2 DNA-binding properties. Surprisingly, CtIP reliably binds to non-canonical DNAends and was found to join DNA molecules. In the third project, the activity of the DNA mismatch repair mechanism in natural competent bacteria is studied. These organisms can introduce and incorporate exogenous DNA into its own DNA material. However, the DNA mismatch repair mechanism regulates and repairs the divergent regions between the two DNA strands. The deactivation of this mechanism increases mutation rate in bacteria. In this work, the role of mismatch repair proteins of competent B. subtilis bacteria is shown for the strand-exchange proces