Cas1-Cas2 complex formation mediates spacer acquisition during CRISPR-Cas adaptive immunity

James K Nuñez; Philip J Kranzusch; Jonas Noeske; Addison V Wright; Christopher W Davies; Jennifer A Doudna

doi:10.1038/nsmb.2820

Cas1-Cas2 complex formation mediates spacer acquisition during CRISPR-Cas adaptive immunity

Nat Struct Mol Biol. 2014 Jun;21(6):528-34. doi: 10.1038/nsmb.2820. Epub 2014 May 4.

Authors

James K Nuñez¹, Philip J Kranzusch², Jonas Noeske¹, Addison V Wright¹, Christopher W Davies¹, Jennifer A Doudna³

Affiliations

¹ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.
² 1] Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA. [2] Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, California, USA.
³ 1] Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA. [2] Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, California, USA. [3] Department of Chemistry, University of California, Berkeley, Berkeley, California, USA. [4] Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.

Abstract

The initial stage of CRISPR-Cas immunity involves the integration of foreign DNA spacer segments into the host genomic CRISPR locus. The nucleases Cas1 and Cas2 are the only proteins conserved among all CRISPR-Cas systems, yet the molecular functions of these proteins during immunity are unknown. Here we show that Cas1 and Cas2 from Escherichia coli form a stable complex that is essential for spacer acquisition and determine the 2.3-Å-resolution crystal structure of the Cas1-Cas2 complex. Mutations that perturb Cas1-Cas2 complex formation disrupt CRISPR DNA recognition and spacer acquisition in vivo. Active site mutants of Cas2, unlike those of Cas1, can still acquire new spacers, thus indicating a nonenzymatic role of Cas2 during immunity. These results reveal the universal roles of Cas1 and Cas2 and suggest a mechanism by which Cas1-Cas2 complexes specify sites of CRISPR spacer integration.

Publication types

Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Adaptive Immunity
CRISPR-Associated Proteins / chemistry
CRISPR-Associated Proteins / metabolism
CRISPR-Associated Proteins / physiology*
CRISPR-Cas Systems*
Clustered Regularly Interspaced Short Palindromic Repeats / physiology*
Crystallography, X-Ray
Endodeoxyribonucleases / chemistry
Endodeoxyribonucleases / metabolism
Endodeoxyribonucleases / physiology*
Endonucleases / chemistry
Endonucleases / metabolism
Endonucleases / physiology*
Escherichia coli / immunology*
Escherichia coli / metabolism
Escherichia coli Proteins / chemistry
Escherichia coli Proteins / metabolism
Escherichia coli Proteins / physiology*
Models, Molecular
Protein Structure, Tertiary

Substances

CRISPR-Associated Proteins
Escherichia coli Proteins
Cas2 protein, E coli
Endodeoxyribonucleases
Endonucleases
YgbT protein, E coli

Associated data

PDB/4P6I

Abstract

Publication types

MeSH terms

Substances

Associated data

Grants and funding