Intrinsic sequence specificity of the Cas1 integrase directs new spacer acquisition.
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.
Biomedical Sciences Research Complex, University of St Andrews, St Andrews, United Kingdom.
Clare
C
Rollie
Faculty of Medicine, Institute of Cell Biology, University of Duisburg-Essen, Essen, Germany.
Stefanie
S
Schneider
School of Life Sciences, Queen's Medical Centre, University of Nottingham, Nottingham, United Kingdom.
Anna Sophie
AS
Brinkmann
School of Life Sciences, Queen's Medical Centre, University of Nottingham, Nottingham, United Kingdom.
Edward L
EL
Bolt
Biomedical Sciences Research Complex, University of St Andrews, St Andrews, United Kingdom.
Malcolm F
http://orcid.org/0000-0003-1543-9342
MF
White
D019843:Archaeal Proteins / Q000378:metabolism*; D064130:CRISPR-Associated Proteins / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000378:metabolism*; D011995:Recombination, Genetic; D013379:Substrate Specificity; D048229:Sulfolobus solfataricus / Q000201:enzymology* / Q000235:genetics*
D019843:Archaeal Proteins; D064130:CRISPR-Associated Proteins; D029968:Escherichia coli Proteins; D004706:Endodeoxyribonucleases; C557650:YgbT protein, E coli
CRISPR; E. coli; Sulfolobus solfataricus; adaptation; biochemistry; integrase