title: Double-stranded endonuclease activity in Bacillus halodurans clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas2 protein.
abstract: 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.
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA.
authors.forename: Ki Hyun
authors.initials: KH
authors.lastname: Nam
authors.forename: Fran
authors.initials: F
authors.lastname: Ding
authors.forename: Charles
authors.initials: C
authors.lastname: Haitjema
authors.forename: Qingqiu
authors.initials: Q
authors.lastname: Huang
authors.forename: Matthew P
authors.initials: MP
authors.lastname: DeLisa
authors.forename: Ailong
authors.initials: A
authors.lastname: Ke
mesh_terms: D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055029:Inverted Repeat Sequences; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D008345:Manganese / Q000737:chemistry / Q000378:metabolism; D016296:Mutagenesis; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D008345:Manganese; D004706:Endodeoxyribonucleases; D008274:Magnesium