Structural and Biochemical Analysis of Nuclease Domain of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated Protein 3 (Cas3)

Sabin Mulepati,Scott Bailey

doi:10.1074/jbc.m111.270017

Abstract

RNA transcribed from clustered regularly interspaced short palindromic repeats (CRISPRs) protects many prokaryotes from invasion by foreign DNA such as viruses, conjugative plasmids, and transposable elements. Cas3 (CRISPR-associated protein 3) is essential for this CRISPR protection and is thought to mediate cleavage of the foreign DNA through its N-terminal histidine-aspartate (HD) domain. We report here the 1.8 Å crystal structure of the HD domain of Cas3 from Thermus thermophilus HB8. Structural and biochemical studies predict that this enzyme binds two metal ions at its active site. We also demonstrate that the single-stranded DNA endonuclease activity of this T. thermophilus domain is activated not by magnesium but by transition metal ions such as manganese and nickel. Structure-guided mutagenesis confirms the importance of the metal-binding residues for the nuclease activity and identifies other active site residues. Overall, these results provide a framework for understanding the role of Cas3 in the CRISPR system.

Highlights

Consistent with this hypothesis, we show that T. thermophilus Cas3HDdom cleaves single-stranded DNA (ssDNA) but not dsDNA
This result establishes that the helicase domain of Cas3 does not alter the substrate specificity of its HD domain
The transition metal ions Mn2ϩ, Co2ϩ, Ni2ϩ, and Zn2ϩ activate the endonuclease activity of T. thermophilus Cas3HDdom but not Mg2ϩ or Ca2ϩ (Fig. 7C)

Summary

EXPERIMENTAL PROCEDURES

Cloning and Mutagenesis—The 780-bp sequence encoding Cas3HDdom was amplified from the Thermus thermophilus HB8 cas gene (TTHB187) and cloned into the pHAT2 expression vector [20] between its NcoI and EcoRI restriction sites. Cell pellets were lysed in lysis buffer (20 mM Tris-HCl, pH 8.0, 1 M NaCl, and 10% glycerol) and clarified by centrifugation at 18,000 rpm for 30 min. The column was washed with lysis buffer containing 20 mM imidazole, and the bound protein was eluted with 250 mM imidazole. The elution was loaded onto a HiLoad 26/60 S200 column (GE Healthcare) pre-equilibrated with gel-filtration buffer (20 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 200 mM NaCl). Fractions containing Cas3HDdom were pooled, dialyzed against gel-filtration buffer lacking EDTA, and concentrated to ϳ8 mg/ml using an Ultracel 10 K centrifugal filter unit (Millipore). Experiments were performed in a buffer containing 20 mM Tris-HCl, pH 8.0, and 200 mM NaCl. The final concentration of protein was 10 ␮M (⑀ ϭ 55460 MϪ1 cmϪ1). Fluorescence intensities were plotted as a function of temperature, and the midpoint of the unfolding transition taken as an estimation of the melting temperature

RESULTS

Heavy atom sites

DISCUSSION

Melt temperature