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The overarching aim of this research programme was to develop novel systems to either transiently or permanently arrest DNA replication forks in eukaryotic cells in a temporally controlled manner. To achieve this, we developed a simple binary system comprising a specific protein that binds with high affinity to a short DNA sequence. This sequence could be engineered into any genome at a defined location and, due to being asymmetric, was shown to impede fork progression in a directional (polar) manner.

The primary focus was on the use of a system adapted from bacteria comprising the protein, Tus, and its recognition sequence, Ter, which together regulate replication termination in E. coli. When inserted into the budding yeast genome, Tus-Ter arrest replication forks in a polar manner, as it does in bacteria, indicating that this polarity is intrinsic to Tus-Ter. Although the replication blockade is eventually overcome in yeast, there are clear ‘signatures’ of permanent genomic alterations that we have characterized. Most notably, a high frequency of mutations arises in a zone behind the point where the replication fork stalls in the already replicated DNA. Most of these mutations arise via the process of homologous recombination and depend upon an unexpected processing of the DNA behind the fork to generate a single-stranded DNA gap via the action of the nuclease, Exonuclease 1. These events are both elevated in frequency and altered in the spectrum of changes in mutants lacking Sgs1, the yeast ortholog of the human BLM protein - which is defective in the cancer predisposition disorder, Bloom’s syndrome. These data have implications for how oncogene-induced replication stress might drive tumorigenesis in humans.

A particularly interesting site for insertion of Ter sites was within a single yeast telomere. This was successfully achieved at two telomeric loci. Replication fork arrest at these chromosome ends elicited many responses in common with those seen at non-telomeric sites (mutagenesis and a Sgs1 effect etc) but also some different responses. The most notable difference was the involvement of two proteins Esc2 and Mph1, in regulating the formation of genomic deletions at the modified telomere, but not elsewhere in the genome. Moreover, esc2 mutant cells generated a recurrent deletion event. Understanding the molecular basis for this mutational ‘hotspot’ is a current priority. Importantly, a ‘toolbox’ of reagents has been generated that is now freely available to the scientific community, and has been distributed to over 10 laboratories in Europe thus far. These include strains with various configurations of Ter sites, cassettes to create new sites of Ter integration and constructs to express Tus tagged in various ways; including, epitopes for commonly used antibodies and green fluorescent protein for protein tracking in live cells.