Disintegration promotes protospacer integration by the Cas1-Cas2 complex

eLife

Makkuni Jayaram¹  Chien-Hui Ma¹  Kamyab Javanmardi¹  Ilya J Finkelstein² 

[1] Department of Molecular Biosciences and Institute of Cell and Molecular Biology, University of Texas at Austin, Austin, United States;Department of Molecular Biosciences and Institute of Cell and Molecular Biology, University of Texas at Austin, Austin, United States;Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, United States;

关键词: CRISPR;    adaptation;    transposition;    E. coli;   

DOI  :  10.7554/eLife.65763

来源: eLife Sciences Publications, Ltd

【 摘 要 】

‘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.

【 授权许可】

CC BY