New single-molecule techniques and their application in the study of DNA break repair
Unrepaired DNA breaks can lead to genomic instability or cell death. They occur frequently during normal cellular metabolism and are caused, for example, by the collapse or stalling of the replication fork in response to DNA damage. Proper DNA-end processing and handling are essential for the survival of the cell and prevention of carcinogenesis. Cells possess robust mechanisms to repair DNA breaks. One such DNA repair mechanism is homologous recombination where the sister chromatid is used as a template for the faithful repair of the DNA break. In Bacteria, this pathway is initiated when a DNA end is processed to a 3-ssDNA overhang terminated at a recombination hotspot (Chi) sequence. This is a substrate for formation of a RecA nucleoprotein filament that catalyses strand exchange to promote repair. Recent data implicate the AddAB helicase-nuclease and the SMC (Structural Maintenance of Chromosomes) complex in the DNA break processing mechanism of the model organism Bacillus subtilis. Interaction between these machines provides a molecular link between DNA dynamics and the initiation of DNA break processing that may co-ordinate replication fork collapse and DNA repair. Single-molecule manipulation and imaging techniques offer huge potential to investigate DNA break repair reactions in completely new ways, providing information that is inaccessible to conventional ensemble experiments. The aim of this project is two-fold: firstly, to develop novel biophysical instruments for fast Atomic Force Microscopy imaging in liquid and a combined Optical and Magnetic Tweezers setup; and secondly, to monitor and characterize the real-time dynamics of these DNA-repair processes using these new and complementary biophysical approaches. Single-molecule investigation will be supported by statistical analysis of the data and conventional bulk biochemical techniques.
Investigador principal: Fernando Moreno Herrero
CENTRO NACIONAL DE BIOTECNOLOGÍA