Moch Clara, Fromant Michel, Blanquet Sylvain, Plateau Pierre
Laboratoire de Biochimie, Ecole polytechnique, CNRS, Université Paris-Saclay, 91128 Palaiseau cedex, France.
Nucleic Acids Res. 2017 Mar 17;45(5):2714-2723. doi: 10.1093/nar/gkw1309.
Prokaryotic adaptive immunity relies on the capture of fragments of invader DNA (protospacers) followed by their recombination at a dedicated acceptor DNA locus. This integrative mechanism, called adaptation, needs both Cas1 and Cas2 proteins. Here, we studied in vitro the binding of an Escherichia coli Cas1-Cas2 complex to various protospacer and acceptor DNA molecules. We show that, to form a long-lived ternary complex containing Cas1-Cas2, the acceptor DNA must carry a CRISPR locus, and the protospacer must not contain 3΄-single-stranded overhangs longer than 5 bases. In addition, the acceptor DNA must be supercoiled. Formation of the ternary complex is synergistic, in such that the binding of Cas1-Cas2 to acceptor DNA is reinforced in the presence of a protospacer. Mutagenesis analysis at the CRISPR locus indicates that the presence in the acceptor plasmid of the palindromic motif found in CRISPR repeats drives stable ternary complex formation. Most of the mutations in this motif are deleterious even if they do not prevent cruciform structure formation. The leader sequence of the CRISPR locus is fully dispensable. These DNA binding specificities of the Cas1-Cas2 integrase are likely to play a major role in the recruitment of this enzyme at the CRISPR locus.
原核生物的适应性免疫依赖于捕获入侵DNA片段（原间隔序列），随后这些片段在特定的受体DNA位点发生重组。这种整合机制，即适应性，需要Cas1和Cas2蛋白。在此，我们在体外研究了大肠杆菌Cas1-Cas2复合物与各种原间隔序列和受体DNA分子的结合。我们发现，为了形成包含Cas1-Cas2的长寿命三元复合物，受体DNA必须携带CRISPR位点，且原间隔序列不能包含长度超过5个碱基的3΄-单链突出端。此外，受体DNA必须是超螺旋的。三元复合物的形成具有协同性，即原间隔序列的存在会增强Cas1-Cas2与受体DNA的结合。对CRISPR位点的诱变分析表明，CRISPR重复序列中发现的回文基序存在于受体质粒中会驱动稳定的三元复合物形成。该基序中的大多数突变即使不阻止十字形结构的形成也是有害的。CRISPR位点的前导序列完全是可有可无的。Cas1-Cas2整合酶的这些DNA结合特异性可能在该酶在CRISPR位点的募集过程中起主要作用。
