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Cas1-Cas2 and DnaK physically and functionally interact to modulate CRISPR Adaptation

ABSTRACT: Prokaryotic Cas1-Cas2 protein complexes generate adaptive immunity to mobile genetic elements (MGEs), by capture and integration of MGE DNA in to CRISPR sites. De novo immunity relies on Cas1-Cas2 targeting MGE DNA, without the aid of pre-existing immunity complexes, through mechanisms of ‘naive adaptation’ that are not clear. Using E. coli we show that the chaperone DnaK inhibits Cas1-Cas2 from DNA binding and integration, and that DnaK expression prevents naïve adaptation from chromosomal self-targeting. We show that that inhibition of naïve adaptation is reversible by eliminating DnaK from cells, by mutation of the DnaK substrate binding domain, and by expression of an MGE (phage )protein. We show that a fluorescently labelled Cas1 fusion can be visualised in living cells. Formation of foci depends on active DNA replication, and that the number of foci per cell is much increased in cells lacking DnaK. We discuss a model in which DnaK provides a mechanism for restraining naïve adaptation from self-targeting. This restraint is released once MGE DNA is present in the cell.

INSTRUMENT(S): LTQ

ORGANISM(S): Escherichia Coli

SUBMITTER: Tom Killelea

LAB HEAD: Edward L. Bolt

PROVIDER: PXD042090 | Pride | 2023-05-18

REPOSITORIES: Pride

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