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
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 stru...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Oxford Journals
2018
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| Online Access: | https://eprints.nottingham.ac.uk/53448/ |
| Summary: | 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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