Double-stranded endonuclease activity in Bacillus halodurans clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas2 protein

J Biol Chem. 2012 Oct 19;287(43):35943-52. doi: 10.1074/jbc.M112.382598. Epub 2012 Aug 31.


The CRISPR (clustered regularly interspaced short palindromic repeats) system is a prokaryotic RNA-based adaptive immune system against extrachromosomal genetic elements. Cas2 is a universally conserved core CRISPR-associated protein required for the acquisition of new spacers for CRISPR adaptation. It was previously characterized as an endoribonuclease with preference for single-stranded (ss)RNA. Here, we show using crystallography, mutagenesis, and isothermal titration calorimetry that the Bacillus halodurans Cas2 (Bha_Cas2) from the subtype I-C/Dvulg CRISPR instead possesses metal-dependent endonuclease activity against double-stranded (ds)DNA. This activity is consistent with its putative function in producing new spacers for insertion into the 5'-end of the CRISPR locus. Mutagenesis and isothermal titration calorimetry studies revealed that a single divalent metal ion (Mg(2+) or Mn(2+)), coordinated by a symmetric Asp pair in the Bha_Cas2 dimer, is involved in the catalysis. We envision that a pH-dependent conformational change switches Cas2 into a metal-binding competent conformation for catalysis. We further propose that the distinct substrate preferences among Cas2 proteins may be determined by the sequence and structure in the β1-α1 loop.

Publication types

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

MeSH terms

  • Bacillus / enzymology*
  • Bacillus / genetics
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Endodeoxyribonucleases / chemistry*
  • Endodeoxyribonucleases / genetics
  • Endodeoxyribonucleases / metabolism
  • Inverted Repeat Sequences
  • Magnesium / chemistry
  • Magnesium / metabolism
  • Manganese / chemistry
  • Manganese / metabolism
  • Mutagenesis
  • Protein Multimerization*
  • Protein Structure, Quaternary
  • Protein Structure, Secondary


  • Bacterial Proteins
  • DNA-Binding Proteins
  • Manganese
  • Endodeoxyribonucleases
  • Magnesium

Associated data

  • PDB/4ES1
  • PDB/4ES2
  • PDB/4ES3