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Abstract
The adaptive prokaryotic immune system CRISPR-Cas provides RNA-mediated protection from invading genetic elements. The fundamental basis of the system is the ability to capture small pieces of foreign DNA for incorporation into the genome at the CRISPR locus, a process known as Adaptation, which is dependent on the Cas1 and Cas2 proteins. We demonstrate that Cas1 catalyses an efficient trans-esterification reaction on branched DNA substrates, which represents the reverse- or disintegration reaction. Cas1 from both Escherichia coli and Sulfolobus solfataricus display sequence specific activity, with a clear preference for the nucleotides flanking the integration site at the leader-repeat 1 boundary of the CRISPR locus. Cas2 is not required for this activity and does not influence the specificity. This suggests that the inherent sequence specificity of Cas1 is a major determinant of the adaptation process.
Highlights
The CRISPR-Cas system is an adaptive immune system present in many archaeal and bacterial species
It was demonstrated that E. coli Cas1 (EcoCas1) cleaved branched DNA substrates preferentially (Babu et al, 2011)
We investigated the activity of S. solfataricus Cas1 (SsoCas1) against a DNA substrate with a 5′-flap structure (Figure 2)
Summary
The CRISPR-Cas system is an adaptive immune system present in many archaeal and bacterial species It provides immunity against viruses and other mobile genetic elements mediated through sequence homology-directed detection and destruction of foreign nucleic acid species (reviewed in Sorek et al, 2013; Barrangou and Marraffini, 2014; van der Oost et al, 2014). The CRISPR locus is transcribed from the leader, generating pre-CRISPR RNA (pre-crRNA) that is subsequently processed into unit length crRNA species and loaded into large ribonucleoprotein complexes. These complexes, known as ‘Interference’, ‘Effector’ or ‘Surveillance’ complexes, utilize crRNA to detect and degrade cognate invading genetic entities, providing immunity against previously encountered viruses and plasmids
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