Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein genes (cas genes) are widespread in bacteria and archaea. They form a line of RNA-based immunity to eradicate invading bacteriophages and malicious plasmids. A key molecular event during this process is the acquisition of new spacers into the CRISPR loci to guide the selective degradation of the matching foreign genetic elements. Csn2 is a Nmeni subtype-specific cas gene required for new spacer acquisition. Here we characterize the Enterococcus faecalis Csn2 protein as a double-stranded (ds-) DNA-binding protein and report its 2.7 Å tetrameric ring structure. The inner circle of the Csn2 tetrameric ring is ∼26 Å wide and populated with conserved lysine residues poised for nonspecific interactions with ds-DNA. Each Csn2 protomer contains an α/β domain and an α-helical domain; significant hinge motion was observed between these two domains. Ca(2+) was located at strategic positions in the oligomerization interface. We further showed that removal of Ca(2+) ions altered the oligomerization state of Csn2, which in turn severely decreased its affinity for ds-DNA. In summary, our results provided the first insight into the function of the Csn2 protein in CRISPR adaptation by revealing that it is a ds-DNA-binding protein functioning at the quaternary structure level and regulated by Ca(2+) ions.
Highlights
Phages, using an RNA-mediated defense mechanism with fundamental similarities to our innate and adaptive immune responses [1,2,3,4,5,6,7]
E. faecalis Csn2 Binds ds-DNA—E. faecalis Csn2 protein recombinantly expressed from E. coli was found to assemble into a large oligomeric state and interact with nucleic acid strongly, displaying higher absorbance at UV260 rather than UV280 after Ni-NTA and size exclusion chromatography (Fig. 1A)
The co-purifying nucleic acids could be extracted from the Csn2 protein using anion exchange chromatography (Fig. 1, B and C) and were shown to be ϳ100 –500 bp in size. They were sensitive to DNase I digestion, but not RNase A digestion nor alkaline hydrolysis treatment that selectively degrades RNA (Fig. 1D), suggesting that the Csn2-bound nucleic acids are likely the E. coli endogenous ds-DNA, but not RNA
Summary
Expression, and Purification—Full-length csn gene (accession number: C7UDU4) from E. faecalis was cloned into a modified pSUMO vector fused with a His6-tagged N-terminal SUMO protein. Resulting Csn proteins were concentrated and further purified on a Superdex 200 column (GE Healthcare) equilibrated with sizing column buffer containing 50 mM Tris-HCl, pH 8.0, 0.2 M NaCl, and 2 mM DTT. Analysis of the Interaction between Csn and ds-DNA—Copurifying nucleic acids were separated from the Csn protein on the Mono Q column, concentrated, and analyzed on a 1% (w/v) agarose gel stained with ethidium bromide. To reveal their identity, DNase I (0.1 g/ml) and RNase A(0.1 g/ml) digestions were carried out in a buffer containing 20 mM HEPES (pH 7.5) for 30 min at 25 °C. Figure illustrations were generated using PyMOL [38]
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