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ERC

REPBLOCK Report Summary

Project ID: 321717
Funded under: FP7-IDEAS-ERC
Country: Denmark

Mid-Term Report Summary - REPBLOCK (SITE-SPECIFIC DNA REPLICATION PERTURBATION AND ITS EFFECTS ON CHROMOSOME SEGREGATION)

The primary goal of ‘Repblock’ was to create novel systems for arresting DNA replication in a site-specific and inducible manner. To achieve this, systems were to be developed whereby sequences recognized by high affinity DNA binding proteins would be integrated into specific loci in eukaryotic genomes. The bulk of this study to date has been conducted in budding yeast, where we have demonstrated that the Tus protein from E.coli when bound to its recognition sequence (Ter) will arrest replication in a polar fashion. This polarity allows great flexibility to be built into the system as we can control the orientation of the Ter sequences relative to defined replication origins.

A key finding of the studies to date is that Tus-mediated replication arrest is both recombinogenic and mutagenic, which has implications for how replication stress in human cancers might drive tumorigenesis. Though extensive genome analysis and DNA sequencing, we are undertaking a functional characterization of the pathways involved in triggering genome alterations.

We have generated yeast stains to permit analysis of Tus-Ter in multiple contexts. The most promising seem to be (i) insertion into a single telomere (ii) creation of a double-blocking configuration that prevents a region of the genome from being replicated normally. This latter system aims to mimic the situation in human cells where replication of common fragile sites is impaired. We also aim to characterize the proteins recruited to the blocking sites in each case using mass spectromertry.

Another key development has been to develop the Tus-Ter system for use in human cells. While this is technically much more demanding than using yeast, we are exploring ways to use sequence modules that can autonomously initiate replication in human cells. In the meantime, we are characterizing an established system for arresting DNA replication in human cells through the use of arrays of Lac operator sites bound by the Lac repressor. This has opened up two new research areas for us in that we find that (i) the LacO/LacR system can induce micronuclei at a high frequency, permitting us to track the fate of these structures in real-time (ii) LacO/R cause activation of cellular senescence within a short time-frame. Having an inducible system for studying DNA damage stress-induced senescence will open up many avenues for research for us.

Reported by

KOBENHAVNS UNIVERSITET
Denmark
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