Journal
NUCLEIC ACIDS RESEARCH
Volume 43, Issue 4, Pages 2216-2222Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkv094
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Funding
- Montana State University (MSU) Undergraduate Research Scholarship
- National Institutes of Health (NIH) [P20GM103500, R01GM108888]
- National Science Foundation EPSCoR [EPS-110134]
- Murdock Charitable Trust
- MSU Agricultural Experimental Station
- NIH IDeA Program COBRE [GM110732]
- MSU Library Open Access Fund
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The Type I-F CRISPR-mediated (clustered regularly interspaced short palindromic repeats) adaptive immune system in Pseudomonas aeruginosa consists of two CRISPR loci and six CRISPR-associated (cas) genes. Foreign DNA surveillance is performed by a complex of Cas proteins (Csy1-4) that assemble with a CRISPR RNA (crRNA) into a 350-kDa ribonucleoprotein called the Csy complex. Here, we show that foreign nucleic acid recognition by the Csy complex proceeds through sequential steps, initiated by detection of two consecutive guanine-cytosine base pairs (G-C/G-C) located adjacent to the complementary DNA target. We show that this motif, called the PAM (protospacer adjacent motif), must be doublestranded and that single-stranded PAMs do not provide significant discriminating power. Binding assays performed with G-C/G-C-rich competitor sequences indicate that the Csy complex interacts directly with this dinucleotide motif, and kinetic analyses reveal that recognition of a G-C/G-C motif is a prerequisite for crRNA-guided binding to a target sequence. Together, these data indicate that the Csy complex first interacts with G-C/G-C base pairs and then samples adjacent target sequences for complementarity to the crRNA guide.
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