Abstract
'Disintegration'-the reversal of transposon DNA integration at a target site-is regarded as an abortive off-pathway reaction. Here, we challenge this view with a biochemical investigation of the mechanism of protospacer insertion, which is mechanistically analogous to DNA transposition, by the Streptococcus pyogenes Cas1-Cas2 complex. In supercoiled target sites, the predominant outcome is the disintegration of one-ended insertions that fail to complete the second integration event. In linear target sites, one-ended insertions far outnumber complete protospacer insertions. The second insertion event is most often accompanied by the disintegration of the first, mediated either by the 3'-hydroxyl exposed during integration or by water. One-ended integration intermediates may mature into complete spacer insertions via DNA repair pathways that are also involved in transposon mobility. We propose that disintegration-promoted integration is functionally important in the adaptive phase of CRISPR-mediated bacterial immunity, and perhaps in other analogous transposition reactions.
Highlights
CRISPR-based bacterial immunity uses information from a prior infection stored in the chromosome to target and destroy invading viruses or plasmids
A fraction of the product migrated as a nicked circle expected for a complete protospacer integration event or semi-integration events at either the L-R or R-S junction (Figure 1C,D)
A split view of the top and bottom portions of the gel is presented to show the plasmid and protospacer bands. (D) Reactions similar to those shown in C utilized a Cas1 to Cas2 molar ratio of 2:1 and were analyzed over a time course. (E) The substrate plasmid used for the reactions in (C) and (D) was treated with a sub-optimal amount of Escherichia coli topoisomerase I to follow the pattern of DNA relaxation over time. (F) The T4 ligase reactions were performed on the plasmid containing the L-R-S target site nicked with Nb.BtsI
Summary
CRISPR-based bacterial immunity uses information from a prior infection stored in the chromosome to target and destroy invading viruses or plasmids. Class 2 systems utilize RNA-guided target recognition and cleavage by a single protein, that is, Cas or Cas. Class 2 systems utilize RNA-guided target recognition and cleavage by a single protein, that is, Cas or Cas12 These two classes are further divided into several types and subtypes (Wright et al, 2016; Makarova et al, 2015; Shmakov et al, 2015). Self-targeting is avoided via multiple mechanisms, including recognition of a protospacer adjacent motif (PAM) immediately bordering the crRNA-complementary DNA sequence (Gasiunas et al, 2012; Jiang and Doudna, 2017; Jinek et al, 2012; Elmore et al, 2016; Pyenson and Marraffini, 2017; Smargon et al, 2017; Wang et al, 2019)
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