Distinct genetic control of homologous recombination repair of Cas9-induced double-strand breaks, nicks and paired nicks
| Autor: | Francesca Cavallo, Maria Jasin, Lianne E.M. Vriend, Chun-Chin Chen, Przemek M. Krawczyk, Fabio Vanoli, Rohit Prakash, Yu Zhang |
|---|---|
| Přispěvatelé: | Other departments, Cell Biology and Histology |
| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
DNA Replication DNA End-Joining Repair DNA damage Genome Integrity Repair and Replication Cell Line 03 medical and health sciences chemistry.chemical_compound Gene Knockout Techniques Mice Genome editing Genetics Animals Humans DNA Breaks Double-Stranded Nucleotide Motifs Homologous Recombination Regulation of gene expression Nuclease biology DNA replication Recombinational DNA Repair Endonucleases Molecular biology enzymes and coenzymes (carbohydrates) 030104 developmental biology chemistry biology.protein Homologous recombination Sister Chromatid Exchange DNA DNA Damage |
| Zdroj: | Nucleic Acids Research Nucleic acids research, 44(11), 5204-5217. Oxford University Press |
| ISSN: | 1362-4962 0305-1048 |
| Popis: | DNA double-strand breaks (DSBs) are known to be powerful inducers of homologous recombination (HR), but single-strand breaks (nicks) have also been shown to trigger HR. Both DSB- and nick-induced HR (nickHR) are exploited in advanced genome-engineering approaches based on the bacterial RNA-guided nuclease Cas9. However, the mechanisms of nickHR are largely unexplored. Here, we applied Cas9 nickases to study nickHR in mammalian cells. We find that nickHR is unaffected by inhibition of major damage signaling kinases and that it is not suppressed by nonhomologous end-joining (NHEJ) components, arguing that nick processing does not require a DSB intermediate to trigger HR. Relative to a single nick, nicking both strands enhances HR, consistent with a DSB intermediate, even when nicks are induced up to ∼1kb apart. Accordingly, HR and NHEJ compete for repair of these paired nicks, but, surprisingly, only when 5' overhangs or blunt ends can be generated. Our study advances the understanding of molecular mechanisms driving nick and paired-nick repair in mammalian cells and clarify phenomena associated with Cas9-mediated genome editing. |
| Databáze: | OpenAIRE |
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