Large Domain Motions in Ago Protein Controlled by the Guide DNA-Strand Seed Region Determine the Ago-DNA-mRNA Complex Recognition Process

Cas9 is a CRISPR-associated endonuclease capable of RNA-guided, site-specific DNA cleavage. The programmable activity of Cas9 has been widely utilized for genome editing applications. Despite extensive studies, the precise mechanism of target DNA binding and on-/off-target discrimination remains incompletely understood. Here we report cryo-EM structures of intermediate binding states of Streptococcus pyogenes Cas9 that reveal domain rearrangements induced by R-loop propagation and PAM-distal duplex positioning. At early stages of binding, the Cas9 REC2 and REC3 domains form a positively charged cleft that accommodates the PAM-distal duplex of the DNA substrate. Target hybridisation past the seed region positions the guide-target heteroduplex into the central binding channel and results in a conformational rearrangement of the REC lobe. Extension of the R-loop to 16 base pairs triggers the relocation of the HNH domain towards the target DNA strand in a catalytically incompetent conformation. The structures indicate that incomplete target strand pairing fails to induce the conformational displacements necessary for nuclease domain activation. Our results establish a structural basis for target DNA-dependent activation of Cas9 that advances our understanding of its off-target activity and will facilitate the development of novel Cas9 variants and guide RNA designs with enhanced specificity and activity.

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Structures of the N-terminal modules imply large domain motions during catalysis by methionine synthase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0308082100 ◽

2004 ◽

Vol 101 (11) ◽

pp. 3729-3736 ◽

Cited By ~ 94

Author(s):

J. C. Evans ◽

D. P. Huddler ◽

M. T. Hilgers ◽

G. Romanchuk ◽

R. G. Matthews ◽

...

Keyword(s):

Methionine Synthase ◽

Large Domain ◽

Domain Motions

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Clutch mechanism of chemomechanical coupling in a DNA resecting motor nuclease

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2023955118 ◽

2021 ◽

Vol 118 (11) ◽

pp. e2023955118

Author(s):

Mihaela-Carmen Unciuleac ◽

Aviv Meir ◽

Chaoyou Xue ◽

Garrett M. Warren ◽

Eric C. Greene ◽

...

Keyword(s):

Single Molecule ◽

Atp Hydrolysis ◽

Protein Domain ◽

Dna Double Strand Breaks ◽

Dna Translocation ◽

Strand Breaks ◽

Chemomechanical Coupling ◽

Clutch Mechanism ◽

Dna Strand ◽

Domain Motions

Mycobacterial AdnAB is a heterodimeric helicase–nuclease that initiates homologous recombination by resecting DNA double-strand breaks (DSBs). The N-terminal motor domain of the AdnB subunit hydrolyzes ATP to drive rapid and processive 3′ to 5′ translocation of AdnAB on the tracking DNA strand. ATP hydrolysis is mechanically productive when oscillating protein domain motions synchronized with the ATPase cycle propel the DNA tracking strand forward by a single-nucleotide step, in what is thought to entail a pawl-and-ratchet–like fashion. By gauging the effects of alanine mutations of the 16 amino acids at the AdnB–DNA interface on DNA-dependent ATP hydrolysis, DNA translocation, and DSB resection in ensemble and single-molecule assays, we gained key insights into which DNA contacts couple ATP hydrolysis to motor activity. The results implicate AdnB Trp325, which intercalates into the tracking strand and stacks on a nucleobase, as the singular essential constituent of the ratchet pawl, without which ATP hydrolysis on ssDNA is mechanically futile. Loss of Thr663 and Thr118 contacts with tracking strand phosphates and of His665 with a nucleobase drastically slows the AdnAB motor during DSB resection. Our findings for AdnAB prompt us to analogize its mechanism to that of an automobile clutch.

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A gate and clamp regulate sequential DNA strand cleavage by CRISPR-Cas12a

10.1101/2021.06.18.448962 ◽

2021 ◽

Author(s):

Mohsin M Naqvi ◽

Laura Lee ◽

Oscar E Torres Montaguth ◽

Mark Szczelkun

Keyword(s):

Dna Recognition ◽

Magnetic Tweezers ◽

Dna Unwinding ◽

Nanopore Sequencing ◽

Loop Formation ◽

Target Strand ◽

Diagnostic Applications ◽

Dna Strand ◽

Domain Motions ◽

Resistant State

CRISPR-Cas12a has been widely used for genome editing and diagnostic applications, yet it is not fully understood how RNA-guided DNA recognition activates the sequential cleavage of the non-target strand (NTS) followed by the target strand (TS). Here we used singlemolecule magnetic tweezers microscopy, ensemble gel-based assays and nanopore sequencing to explore the coupling of DNA unwinding and cleavage. In addition to dynamic R-loop formation, we also directly observed transient dsDNA unwinding downstream of the 20 bp DNA:RNA hybrid and, following NTS cleavage and prior to TS cleavage, formation of a hyperstable "clamped" Cas12a-DNA intermediate resistant to DNA twisting. Alanine substitution of a conserved aromatic amino acid "gate" in the REC2 domain that normally caps the heteroduplex produced more frequent and extended downstream DNA breathing, a longer-lived twist-resistant state, and a 16-fold faster rate of TS cleavage. We suggest that both breathing and clamping events, regulated by the gate and by NTS cleavage, deliver the unwound TS to the RuvC nuclease and result from previously described REC2 and NUC domain motions.

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