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Nat Methods. 2009 Oct;6(10):753-7. doi: 10.1038/nmeth.1372. Epub 2009 Sep 13.

A Flp-nick system to study repair of a single protein-bound nick in vivo.

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1
Department of Molecular Biology, University of Aarhus, Aarhus, Denmark.

Abstract

We present the Flp-nick system, which allows introduction of a protein-bound nick at a single genomic site in Saccharomyces cerevisiae and thus mimics a stabilized topoisomerase I-DNA cleavage complex. We took advantage of a mutant Flp recombinase that can introduce a nick at a specific Flp recombinase recognition target site that has been integrated in the yeast genome. The genetic requirement for cells to cope with this insult is the same as for cells treated with camptothecin, which traps topoisomerase I-DNA cleavage complexes genome-wide. Hence, a single protein-bound nick is enough to kill cells if functional repair pathways are lacking. The Flp-nick system can be used to dissect repair, checkpoint and replication fork management pathways activated by a single genomic insult, and it allows the study of events at the damage site, which so far has been impossible to address.

PMID:
19749762
DOI:
10.1038/nmeth.1372
[Indexed for MEDLINE]
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