Objetivo DNA polymerases (DNA Pols) make various decisions during DNA replication. Decisions regarding nucleotide selection, lesion bypass and how to respond to replication errors are a few important examples. During replication, DNA Pols can synthesize DNA at remarkable rates, e.g. 1,000 bp/s for E. Coli DNA Pol III. However, errors can be introduced during replication, resulting in a terminal DNA mismatch. Mismatches can then be sensed and removed by action of the 3’ to 5’ exonuclease activity of the DNA Pol. How DNA Pols can sense mismatches to initiate proofreading and how the primer strand migrates into the exonuclease domain, often a distance of 30 Å, is the focus of this proposal. Moreover, this research aims to understand mechanisms of proofreading in higher complexity DNA Pol assemblies at single-molecule resolution. Proofreading will be studied with the model enzyme E. Coli DNA Pol III, consisting of separate protein subunits that assemble to form a higher order complex, namely the polymerase, exonuclease and the β2-clamp, which is a processivity factor that encircles the DNA and increases the affinity of the polymerase to the DNA. The proposed work will employ single-molecule FRET in order to evaluate proofreading dynamics at high spatial and temporal resolution. Three main objectives will be performed in order to understand (1) how DNA mismatches initiate proofreading (2) how mutations within the exonuclease domain affect proofreading and (3) how DNA lesions can influence proofreading as a function of position within the DNA template. These objectives will be achieved with a single-molecule FRET assay containing fluorescently labelled DNA to monitor proofreading dynamics in real time. This work will shift the knowledge frontier by advancing our understanding of proofreading during DNA replication in order to better realise the relationship between DNA mutations and the development of human diseases like cancer. Ámbito científico natural sciencesbiological sciencesgeneticsDNAnatural sciencesbiological sciencesgeneticsmutationmedical and health sciencesclinical medicineoncologynatural scienceschemical sciencesorganic chemistryaminesnatural sciencesbiological sciencesbiochemistrybiomoleculesproteinsenzymes Palabras clave DNA polymerase DNA replication DNA proofreading Single-molecule imaging Fluorescence resonance energy transfer Programa(s) H2020-EU.1.3. - EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions Main Programme H2020-EU.1.3.2. - Nurturing excellence by means of cross-border and cross-sector mobility Tema(s) MSCA-IF-2015-EF - Marie Skłodowska-Curie Individual Fellowships (IF-EF) Convocatoria de propuestas H2020-MSCA-IF-2015 Consulte otros proyectos de esta convocatoria Régimen de financiación MSCA-IF - Marie Skłodowska-Curie Individual Fellowships (IF) Coordinador IMPERIAL COLLEGE OF SCIENCE TECHNOLOGY AND MEDICINE Aportación neta de la UEn € 183 454,80 Dirección SOUTH KENSINGTON CAMPUS EXHIBITION ROAD SW7 2AZ LONDON Reino Unido Ver en el mapa Región London Inner London — West Westminster Tipo de actividad Higher or Secondary Education Establishments Enlaces Contactar con la organización Opens in new window Sitio web Opens in new window Participación en los programas de I+D de la UE Opens in new window Red de colaboración de HORIZON Opens in new window Coste total € 183 454,80
