NEW YORK (GenomeWeb) – A research team led by the University of California, Berkeley's Jennifer Doudna has discovered that the Cas1-Cas2 complex interacts with DNA based on its own structural features, rather than through direct recognition of the DNA sequence.
While researchers have previously shown that bacteria can use the CRISPR-Cas system to develop adaptive immunity, the primary collaboration between the Cas1 and Cas2 enzymes is less well understood.
As they reported today in Science, the researchers found that the CRISPR-Cas1-Cas2 integrase indirectly recognizes DNA based on deformation of the repeat and flanking sequences, instead of directly recognizing the sequence.
Doudna and her team first presented crystal structures of Cas1-Cas2 bound to both donor and target DNA in intermediate and product integration complexes in order to determine how Cas1-Cas2 recognizes its target sequence. By designing the full-sight product with a break in the middle of protospacer, they allowed the Cas1-Cas2 complex to access the repeat on the sequence. Charge-swap mutations of residues on the enzymes reduce or eliminate acquisition of new spacers, confirming their importance for the CRISPR integration reaction.
Type 1 Cas1-Cas2 from Escherichia coli relies heavily on active site positioning and structure features of the DNA, rather than direct sequence recognition, to localize DNA integration to the CRISPR locus, the authors wrote. The CRISPR-Cas system then depends on the Cas1-Cas2 integrase to capture and integrate short foreign DNA fragments into the CRISPR locus, enabling adaptation to new viruses.
"The DNA substrate duplex's ability to access both Cas1 active sites regulates recognition of the CRISPR repeat," they noted. "The GC-rich inverted repeats allow for twist deformation while the mid-repeat sequence acts a hinge."
In order to figure out the mechanism by which the accessory protein Integration Host Factor (IHF) recruits Cas1-Cas2 to the leader repeat, researchers performed an in vitro integration assay with targets containing leaders with mutations in the upstream binding region or leaders truncated prior to the upstream region. They found that nonspecific interactions with the leader DNA are crucial for Cas1-Cas2 activity and specificity. Further investigating IHF's role in the replication process, the team then performed integration assays with and without IHF. The results demonstrated that IHF aids in the recognition of the target DNA, highlighting the shift in substrate selectivity from donor to target DNA.
The researchers found that when IHF binding distorts the DNA structure at nearly a 30 degree bend, IHF increases both its efficiency and specificity of integration. This action positions the DNA in close proximity to two active sites within the Cas1-Cas2 enzymes.
These results explain how the Cas1-Cas2 CRISPR integrase recognizes a sequence-dependent DNA structure to guarantee site-selective CRISPR array expansion during the first step of bacterial adaptive immunity.
"The unique substrate preferences of the CRISPR integrase could make it useful as a molecular recording device for barcoding genomes" the authors wrote. "Changes in integrase could thereby be exploited for genome tagging applications."