Seminar “Quantifying the physics inside the genome one molecule at a time”

Date

Wednesday, 10th April 2013

Time

12:00 am

Place

University of Barcelona
Faculty of Physics Building
Room 3.20, 3rd floor 

Speaker

Dr Gijs Wuite, Vrije Universiteit Amsterdam (The Netherlands)

Abstract

Homologous recombination is essential for the preservation of genome stability. The core protein in this process, RAD51, drives homology search and DNA strand exchange, processes that requires the nucleation, assembly and disassembly (collapse) of a RAD51 filament on single-stranded (ss) and double-stranded (ds)DNA, coupled to ATP binding and hydrolysis. Here we show that we can characterize all these RAD51 DNA transactions on long individual DNA molecules, in real-time, at the single-protein level using a combination of single-molecule fluorescence microscopy and optical tweezers.

These experiments show that the sizes of RAD51 nuclei on ssDNA vary and display a broad Poissonian distribution with an average size of 4 monomers. Filament extension tracked in time with single-protein resolution reveals that nuclei extend by one RAD51 monomer at a time with a rate independent of tension on the ssDNA. This is in contrast to force-dependent monomeric extension on dsDNA. Counting and timing individual RAD51 monomers disassembling from nucleoprotein filament on ssDNA also yields contrasting results compared with dsDNA, reflecting the difference in the underlying mechanical properties ssDNA and dsDNA based nucleoprotein filaments. Together, these results yield unprecedented quantitative insight in the mechanical rearrangement during formation and collapse of RAD51 nucleoprotein filaments.