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Killelea, Tom and Radovcic, Marin and Savitskaya, Ekaterina and Ivančić-Baće, Ivana and Bolt, Edward L. (2018) 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. Nucleic Acids Research . ISSN 0305-1048 (In Press)

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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 E. coli, DNA integration catalysed 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.

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