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CRISPR-Cas adaptation in Escherichia coli requires RecBCD helicase but not nuclease activity, is independent of homologous recombination, and is antagonised by 5’ ssDNA exonucleases

Radovčić, Marin; Killelea, Tom; Savitskaya, Ekaterina; Wettstein, Lukas; Bolt, Edward L.; Ivančić-Baće, Ivana

Authors

Marin Radovčić

Tom Killelea

Ekaterina Savitskaya

Lukas Wettstein

Edward L. Bolt

Ivana Ivančić-Baće

Abstract

Prokaryotic adaptive immunity is established against mobile genetic elements (MGEs) by ‘naïve adaptation’ when DNA fragments from a newly encountered MGE are integrated into CRISPR–Cas systems. In Escherichia coli, DNA integration catalyzed by Cas1–Cas2 integrase is well understood in mechanistic and structural detail but much less is known about events prior to integration that generate DNA for capture by Cas1–Cas2. Naïve adaptation in E. coli is thought to depend on the DNA helicase-nuclease RecBCD for generating DNA fragments for capture by Cas1–Cas2. The genetics presented here show that naïve adaptation does not require RecBCD nuclease activity but that helicase activity may be important. RecA loading by RecBCD inhibits adaptation explaining previously observed adaptation phenotypes that implicated RecBCD nuclease activity. Genetic analysis of other E. coli nucleases and naïve adaptation revealed that 5′ ssDNA tailed DNA molecules promote new spacer acquisition. We show that purified E. coli Cas1–Cas2 complex binds to and nicks 5′ ssDNA tailed duplexes and propose that E. coli Cas1–Cas2 nuclease activity on such DNA structures supports naïve adaptation.

Journal Article Type Article
Publication Date Nov 2, 2018
Journal Nucleic Acids Research
Print ISSN 0305-1048
Electronic ISSN 1362-4962
Publisher Oxford University Press (OUP)
Peer Reviewed Peer Reviewed
Volume 46
Issue 19
Pages 10173–10183
DOI https://doi.org/10.1093/nar/gky799
Keywords Genome integrity; Repair and replication
Publisher URL https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gky799/5090770

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