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

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    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.

    OriginalsprogEngelsk
    TitelMethods in Enzymology : Intersection between Homologous Recombination, DNA Replication and DNA Repair
    RedaktørerMarie Spies, Anna Malkova
    Antal sider25
    Vol/bind601
    ForlagElsevier
    Publikationsdato3 feb. 2018
    Sider1-25
    Kapitel1
    ISBN (Trykt)978-0-12-813979-0
    DOI
    StatusUdgivet - 3 feb. 2018
    NavnMethods in Enzymology
    ISSN0076-6879

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