Repetitive DNA reeling by the Cascade-Cas3 complex in nucleotide unwinding steps

Abstract

Prokaryotes mediate defense against invading genetic elements using RNA guided adaptive immune systems that are encoded by CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) loci 1,2 . In the type I system, the most ubiquitous CRISPR-Cas system 3 , foreign DNA targets (called protospacers) are recognized by the CRISPR RNA (crRNA)-guided surveillance complex Cascade 4 . Recognition of double stranded DNA targets results in the formation of an R-loop, in which the crRNA hybridizes with the complementary target strand and the non-complementary strand of the DNA is displaced (nontarget strand) [5][6][7][8][9][10] . This R-loop formation triggers a conformational change in the Cascade complex 6,10-12 and leads to the recruitment of the Cas3 protein for subsequent target degradation [13][14][15] .The E. coli Cas3 protein consists of two domains: a N-terminal metal-dependent histidine-aspartate (HD) nuclease domain and a C-terminal superfamily 2 helicase domain 3,13,[16][17][18][19] . Cas3 is activated by the Cascade-marked R-loop, where it cleaves the displaced nontarget strand ~11 nucleotides into the R-loop region 16,20 . Driven by ATP, Cas3 then moves along the nontarget strand in a 3' to 5' direction, while catalyzing cobalt-dependent DNA degradation 14,16,20,21 . Subsequently, Cas3 generates degradation products that are close to spacer length and enriched for PAM-like NTT sequences in their 3' ends 22 . This makes a considerable fraction of the degradation products suitable substrates for integration by the Cas1-Cas2 integrases into the CRISPR locus 22,23 . Even though the biochemistry of CRISPR interference has been largely covered, the biophysics of DNA unwinding by Cas3 remains elusive. In particular, how the helicase domain tunes the property of nuclease HD domain for spacer integration and how this process takes place in concert with Cascade. Results Single-molecule observation of DNA reeling by Cas3We set out to understand how Cas3 unwinds dsDNA substrates. To date, two models prevail for DNA unwinding by the Cas3 helicase: a translocation model and a reeling model. In the translocation model, Cas3 breaks its contacts with the Cascade complex while unwinding the DNA. Thereby Cas3 translocates away from the Cascade binding site and degrades single-stranded DNA fragments along the way (Fig 1a) [13][14][15][16]21,24 . In the reeling model, Cas3 and Cascade remain in tight contact while Cas3 unwinds the DNA, which may result in loops in the target strand (Fig 1a) 21,25 . To distinguish between these two models, we sought to visualize the DNA unwinding activity of Cas3 with a high spatiotemporal resolution.To visualize DNA unwinding by Cas3, we developed an assay based on single-molecule Förster resonance energy transfer (smFRET). In brief, anti-maltose binding protein (MBP) antibodies were anchored to the surface of a polyethylene glycol (PEG)-coated slide through biotinstreptavidin linkage followed by tethering of MBP-fused Cas3 monomers (Fig. 1b & Extended Data Fig. 1a...

Cas3 Mediated Target DNA Recognition and Cleavage is Independent of the Composition and Architecture of Cascade Surveillance Complex

“…This appears to be an ingenious strategy to time the cleavage. Our data on Cas3/I-C are in agreement with the recent finding where stalling of Cas3/I-E translocation at roadblocks stimulates nuclease activity which is otherwise reported to be sparse (49,67). After the Cas3/I-C loading onto the single-stranded region, we envisage two modes of helicase motor activity: (i) translocation -Cas3/I-C is mobile with respect to the bound ssDNA and (ii) reeling -Cas3/I-C is stationary with respect to the bound ssDNA.…”

“…However, our data suggest that upon recruitment to the target site, Cas3/I-C nicks non-target strand at ~30-35 nt from PAM ( Figure 5). This initial nick in the absence of ATP points towards the direct binding of nuclease domain at PAM distal region of R-loop, which then renders a loading point for the helicase domain (49). This also concurs with the interpretation from the recent Cryo-EM structure of Cascade/I-E and Cas3/I-E (46).…”

“…In tune with this, the domain architecture of Cas3 in some sub-types has evolved in a very distinctive way such that HD and Helicase domains, which are fused in some sub-types, have undergone domain fission in others (44,47,48). The current mechanistic understanding on CRISPR interference in type I stems majorly from the prototypical type I-E system (24,30,33,36,39,41,42,46,49,50) with sporadic investigations from other type I systems such as I-A, I-F and I-G (47,48,(51)(52)(53)(54)(55)(56)(57)(58). In type I-C, the Cascade complex is composed of only three subunits, viz.…”

Selective Prespacer Processing Ensures Precise CRISPR-Cas Adaptation

“…We hypothesize that this complex formation allows direct transfer of the ssDNA fragments to Cas1-Cas2, ensuring robust primed spacer acquisition. Recent single-molecule studies have shown that Cas3 generates ssDNA loops during CRISPR interference (Dillard et al, 2018;Loeff et al, 2018). It will be of interest to find out whether this ssDNA loop marked with PAM sequences can be directly recognized by the Cas1-Cas2 complex.…”

“…The fluorescent label Cy3 and Cy5 were imaged using prism-type total internal reflection microscopy (Loeff et al, 2018). In brief, Cy3 was imaged through excitation by a 532 nm diode laser (Compass 215M-50, Coherent).…”