Disintegration promotes protospacer integration by the Cas1-Cas2 complex

Elife. 2021 Aug 26;10:e65763. doi: 10.7554/eLife.65763.

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.

Keywords: CRISPR; E. coli; adaptation; biochemistry; chemical biology; transposition.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism
  • CRISPR-Associated Proteins / genetics*
  • CRISPR-Associated Proteins / metabolism
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
  • Endonucleases / genetics*
  • Endonucleases / metabolism
  • Streptococcus pyogenes / genetics*
  • Streptococcus pyogenes / metabolism

Substances

  • Bacterial Proteins
  • CRISPR-Associated Proteins
  • Endonucleases