Using the Flp Recombinase to Induce Site-Specific Protein-DNA Nicks.
Methods Enzymol
; 601: 1-25, 2018.
Article
em En
| MEDLINE
| ID: mdl-29523229
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.
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MEDLINE
Assunto principal:
Saccharomyces cerevisiae
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Técnicas Genéticas
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DNA Nucleotidiltransferases
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Quebras de DNA de Cadeia Simples
Idioma:
En
Ano de publicação:
2018
Tipo de documento:
Article