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ERC

SMI-DDR Report Summary

Project ID: 268788
Funded under: FP7-IDEAS-ERC
Country: United Kingdom

Final Report Summary - SMI-DDR (Single Molecule Imaging of the DNA Damage Response in Live Cells)

Replication errors underpin the genetic instability of cancer cells. The aim of the program was to further understand what happens when replication forks arrest and the mechanisms by which they restart. We proposed to apply super-resolution and single molecule plus single cell imaging methods to elucidate the behavior of proteins involved in these processes. Using fission yeast as a model system we first developed tools to examine specific genomic regions that are replicated by forks that have been restarted by homologous replication. We also developed a novel deep-sequencing method to examine replication polymerase usage across the genome. We established and applied several novel microscopy methodologies to further understand replication fork arrest and restart. In addition to developing a photo-activation localization microscope (PALM) and an associated suite of analytical software, a novel variation of motion-blur PALM was developed that allowed us to quantify, in individual live cells, the chromatin association of replication and repair factors. We have exploited this method to characterize the dynamics of a variety of DNA replication protein in response to replication arrest and restart. An additional development was an adaptation of fluorescence correlation spectroscopy (FCS) for photo-activation (PA): we have established a protocol to visualize diffusion constants of highly expressed proteins using PA-FCS. Using a combination of these methods and classical molecular and cell biology tools, our main achievements have been the demonstration that HR-restarted replication forks are highly error prone and that both the leading and lagging strands are replicated by polymerase delta.

Reported by

UNIVERSITY OF SUSSEX
United Kingdom
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