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.
Highlights
CRISPR–Cas is a prokaryotic adaptive immune system against mobile genetic elements (MGEs) in bacteria and archaea [1,2]
‘Naıve adaptation’ relies on Cas1– Cas2 for cells to establish new immunity against an MGE that has not been previously encountered by integration of new spacer DNA into CRISPR arrays [4]
These results are in agreement with a model in which RecBCD nuclease activity is important for naive adaptation in E. coli [21] because neither recB or recD cells possess RecBCD nuclease activity
Summary
CRISPR–Cas is a prokaryotic adaptive immune system against mobile genetic elements (MGEs) in bacteria and archaea [1,2]. Immunity is acquired through capture of MGE DNA fragments (‘protospacers’) and their site-specific integration into a CRISPR array as ‘spacers’ positioned between repeat DNA sequences. These processes are called adaptation and are catalysed by Cas1–Cas integrase from host CRISPR–Cas systems aided by other host proteins, reviewed recently in [3]. ‘Naıve adaptation’ relies on Cas1– Cas for cells to establish new immunity against an MGE that has not been previously encountered by integration of new spacer DNA into CRISPR arrays [4]. In Escherichia coli, interference R-loops are formed by Cascade (CRISPR-associated complex for antiviral defence) after detecting MGE DNA through a protospacer adjacent motif (PAM) sequence [5,6]. Cascade Rloop formation recruits Cas nuclease/helicase for degradation of the MGE DNA completing the immunity response [7,8,9]
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