Using the Flp Recombinase to Induce Site-Specific Protein-DNA Nicks

    Research output: Contribution to book/anthology/report/proceedingBook chapterResearchpeer-review

    Abstract

    A natural and frequent occurring replication insult is generated by the action of DNA Topoisomerase I (Top1). When Top1 gets trapped in a cleavage complex on the DNA, a protein-linked DNA nick (PDN) is generated. Today it is known that PDNs are generated at a high incidence in the cell. If not rapidly removed, PDNs can have a profound impact on cell destiny, as a nick in proliferating cells is passively transformed into a single-ended DSB, when encountered by the replication machinery. A DSB can in turn lead to chromosomal rearrangements and thus jeopardize genome stability if not appropriately repaired. In order to study repair pathways associated with PDNs, we have developed a cellular system (Flp-nick), where we can generate a single PDN at a specific genomic site in the model organism Saccharomyces cerevisiae. The system takes advantages of the Flp recombinase, which catalytically operates like Top1 by generating a nick in the DNA backbone and during this process becomes covalently linked to the DNA. Flp cleaves at well-defined target sites. Thus, a target site has been inserted in the genome and a mutant Flp, which cleaves but do not religate, is expressed. In this way, a single PDN mimicking the one generated by Top1 is induced at a known genomic site. The Flp-nick system allows detailed molecular analysis of repair pathways associated with this type of damage and can be designed to study repair at any genomic context.

    Original languageEnglish
    Title of host publicationMethods in Enzymology : Intersection between Homologous Recombination, DNA Replication and DNA Repair
    EditorsMarie Spies, Anna Malkova
    Number of pages25
    Volume601
    PublisherElsevier
    Publication date3 Feb 2018
    Pages1-25
    Chapter1
    ISBN (Print)978-0-12-813979-0
    DOIs
    Publication statusPublished - 3 Feb 2018
    SeriesMethods in Enzymology
    ISSN0076-6879

    Keywords

    • Break-induced replication
    • Double-strand break repair
    • Nick repair
    • Protein–DNA adduct
    • Replication damage
    • Topoisomerase I

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