Cas3 of the Pseudomonas aeruginosa UCBPP-PA14 type I-F (Yersinia-subtype) CRISPR-Cas system is a 1076-residue Cas2-Cas3 fusion protein: type I-F systems encode no separate cas2 gene, so a Cas2 module occupies the N-terminal approximately 120 residues ahead of an HD-family metal-dependent nuclease domain and a superfamily 2 ATP-dependent helicase domain. The two halves serve the two halves of the pathway. Through its Cas2 module the protein is required for capture and integration of new spacers alongside Cas1, and through its nuclease and helicase domains it is the destruction machine of the interference stage: once the Csy surveillance complex has formed a PAM-licensed R-loop on an invading DNA, Cas3 is recruited to the C-terminal helical bundle of the Csy large subunit Cas8f and processively unwinds and degrades the target. Point substitutions in either the nuclease or the helicase catalytic centre abolish function in vivo. The recruitment step is the sole target of the phage anti-CRISPR protein AcrIF3, which clamps Cas3 in a nucleotide-bound state, occludes the helicase DNA-binding tunnel and masks the surface the Csy complex uses.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0043571 maintenance of CRISPR repeat elements | IMP PMID:26586803 Foreign DNA acquisition by the I-F CRISPR-Cas system require... | ACCEPT | Summary: Well grounded and correctly hedged. Three independent lesions in this gene abolish new spacer acquisition in the transplanted Pseudomonas aeruginosa type I-F system: a stop codon placed after the Cas2 module, a nuclease domain D124A substitution, and a helicase domain D576N substitution. The acts_upstream_of_or_within qualifier is right because in type I-F the acquisition requirement is distributed across the fused Cas2 module, the catalytic domains, and the Csy complex, rather than resting on a single Cas3 step in the integration chemistry. Supporting Evidence: PMID:26586803 A D124Oc mutation in the cas2+cas3 gene that introduced an ochre stop codon instead of aspartate codon at position 124, after the cas2 portion of the fused gene, abolished spacer acquisition PMID:26586803 Point mutations introducing single amino acid substitutions in the endonuclease (D124A) and helicase (D576N) domains of the Cas3 protein also prevented spacer acquisition PMID:26586803 A specific feature of type I-F systems is a fusion of cas2 and cas3 homologs, which are encoded on separate genes in other CRISPR |
| GO:0004536 DNA nuclease activity | IDA PMID:32170016 Inhibition mechanisms of AcrF9, AcrF8, and AcrF6 against typ... | NEW | Summary: Cas3 is the entity that degrades the target DNA. Reconstitution of the Pseudomonas aeruginosa Csy complex with Cas3, ATP and Mg(2+) gives cleavage of a labelled double-stranded DNA substrate that is strictly Cas3-dependent: the no-Cas3 lane is the negative control, and relieving anti-CRISPR inhibition restores cleavage. Reason: GOA for Q02ML8 holds only the two CACAO rows on GO:0043571 and no molecular function at all, not even an IEA, which is almost certainly because the fused Cas2-3 architecture does not match the signatures the electronic pipelines key on. Comparator check: the Escherichia coli orthologue Cas3 (P38036) carries GO:0000014, GO:0008296, GO:0033677, GO:0003690, GO:0000287 and GO:0003724, and every type I system's Cas3 is the degradation nuclease, so a blank molecular-function profile here is a one-protein pipeline failure rather than an ontology convention. I deliberately propose the moderately specific GO:0004536 rather than the Escherichia coli children GO:0000014 or GO:0008296: the assay used double-stranded DNA presented by the Csy complex and did not resolve exonucleolytic versus endonucleolytic mode or strand preference for this orthologue, so asserting those children would transfer a result that cannot be cited for this protein. Supporting Evidence: PMID:32170016 WT AcrF9 inhibits the DNA binding and subsequent Cas3 recruitment of the Csy complex, suppressing DNA degradation PMID:32170016 We found that these mutations result in much stronger DNA cleavage activities of Cas3 compared to the WT AcrF6 PMID:32170016 The Cas3 protein (P. aeruginosa) was proposed to interact with the C-terminal helical bundle of Cas8f |
| GO:0004386 helicase activity | IMP PMID:26586803 Foreign DNA acquisition by the I-F CRISPR-Cas system require... | NEW | Summary: The protein carries a superfamily 2 helicase domain with a DEAD-box motif at residues 576 to 579, substitution of the motif aspartate abolishes function in vivo in two independent assays, and the crystal structure of this protein shows the helicase domain in a nucleotide-bound state with a DNA-binding tunnel that an inhibitor can plug. Reason: Missing from GOA along with every other molecular function for this protein. Again the generic term rather than the Escherichia coli orthologue's GO:0033677 DNA/RNA helicase activity: no unwinding assay on Pseudomonas aeruginosa Cas3 has established which duplex it acts on, so the substrate-specific child is not supportable here. The evidence code is IMP because the grounding is catalytic-residue mutants rather than a direct unwinding measurement. Supporting Evidence: PMID:26586803 Point mutations introducing single amino acid substitutions in the endonuclease (D124A) and helicase (D576N) domains of the Cas3 protein also prevented spacer acquisition PMID:21398535 the core Cas protein Cas3 functions downstream of small crRNA production and that this protein requires functional HD (predicted phosphohydrolase) and DEXD/H (predicted helicase) domains to suppress biofilm formation in DMS3 lysogens PMID:27455460 locks PaCas3 in an ADP-bound form, blocks the entrance of the DNA-binding tunnel in the helicase domain, and masks the linker region and C-terminal domain of PaCas3, thereby preventing recruitment by Cascade and inhibiting the type I-F CRISPR-Cas system |
| GO:0005524 ATP binding | IDA PMID:27455460 Structural basis of Cas3 inhibition by the bacteriophage pro... | NEW | Summary: This protein was crystallised with an adenine nucleotide bound in its helicase domain, which the anti-CRISPR protein AcrF3 exploits by locking the ADP-bound state. The in vitro DNA cleavage assays are likewise run with ATP in the reaction. Reason: Missing from GOA; the Escherichia coli orthologue carries GO:0005524 by IEA, while here there is a direct structural observation on the protein itself. Kept as a supporting rather than core function. Supporting Evidence: PMID:27455460 we report the crystal structure of the anti-CRISPR protein AcrF3 in complex with Pseudomonas aeruginosa Cas3 (PaCas3) PMID:27455460 locks PaCas3 in an ADP-bound form, blocks the entrance of the DNA-binding tunnel in the helicase domain, and masks the linker region and C-terminal domain of PaCas3, thereby preventing recruitment by Cascade and inhibiting the type I-F CRISPR-Cas system |
| GO:0099048 CRISPR-cas system | IMP PMID:26586803 Foreign DNA acquisition by the I-F CRISPR-Cas system require... | NEW | Summary: Cas3 performs the target interference stage that this term's definition names as the third of the system's three stages, and through its Cas2 module it also acts in the acquisition stage. Both are supported by gene-specific mutant phenotypes in this strain's system. Reason: Comparator check: every Escherichia coli Cascade subunit plus Cas1 and Cas2 carry involved_in GO:0099048 by IDA, yet no Pseudomonas aeruginosa PA14 cas gene does, which marks a per-strain curation lag rather than a convention. Checked against QuickGO: GO:0099048 is neither an ancestor nor a descendant of GO:0043571, so it does not duplicate the existing annotation. Cas3 itself does the unwinding and degradation, so this is participation, not mere requirement. Supporting Evidence: PMID:26586803 Point mutations introducing single amino acid substitutions in the endonuclease (D124A) and helicase (D576N) domains of the Cas3 protein also prevented spacer acquisition PMID:28985564 Formation of the R-loop in both Csy and the related Type I-E Cse complexes triggers recruitment of the endonuclease Cas3, which degrades the target dsDNA PMID:26416740 The third anti-CRISPR protein operates by binding to the Cas3 helicase-nuclease and preventing its recruitment to the DNA-bound CRISPR-Cas complex |
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Download this section (compressed HTML)Q: Does the Cas2 module of PA14 Cas2-3 contribute anything to interference, or is its role confined to adaptation? The D124 position sits at the module boundary and is also the HD nuclease metal ligand in the numbering used by the mutagenesis studies, so the two functions are hard to separate genetically.
Q: AcrIF3 blocks Cas3 recruitment while leaving the Csy complex able to bind its target, which converts the system into a sequence-specific DNA-binding module. Should that gain-of-function state (reported as conversion into a transcriptional repressor) be captured in GO at all, given that it occurs only during phage infection?
Q: Is PA14 Cas3 recruited as a monomer or as an oligomer, and does the nucleotide state that AcrIF3 traps correspond to the recruitment-competent conformation or to a post-translocation one?
Experiment: Reconstitute the Csy complex with a target plasmid carrying strand-specific labels and run time-course cleavage with purified PA14 Cas2-3, mapping products by denaturing gel and sequencing. Repeat with HD-dead (D124A) and helicase-dead (D576N) proteins to assign products to each catalytic centre.
Hypothesis: PA14 Cas3 degrades the displaced non-target strand first and processively, as the Escherichia coli orthologue does, rather than cleaving both strands at the R-loop.
Type: in vitro nuclease mapping
Experiment: Measure ATPase and unwinding of defined forked substrates by purified Cas2-3 alone and in the presence of the Csy complex, and test whether AcrIF3 inhibits the intrinsic activity or only the Cas8f-dependent stimulation.
Hypothesis: The helicase domain of PA14 Cas3 is an ATP-dependent 3' to 5' DNA translocase whose activity is licensed by Cas8f contact.
Type: single-molecule or ensemble helicase assay
What is not known — curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: No nuclease or helicase activity has been measured on purified Pseudomonas aeruginosa Cas3 in isolation, so the mode of DNA cleavage (endonucleolytic versus exonucleolytic, strand preference, processivity) and the duplex substrate of the helicase are undetermined for this orthologue.
OPEN BIOLOGYCURATION
What is known: What is established is that Cas3 is necessary and sufficient to convert Csy-bound DNA into degraded product in vitro, that both catalytic centres are required in vivo, and that the helicase domain binds nucleotide and has a DNA-binding tunnel. The Escherichia coli orthologue's detailed activities (single-stranded DNA endonuclease, 3'-5' exonuclease, DNA/RNA helicase) have not been reproduced on this protein.
Significance: The absence of any molecular-function annotation on Q02ML8, in contrast to nine on the Escherichia coli orthologue, makes the destruction step of the best-studied anti-CRISPR system invisible to GO-based reasoning, and leaves the nature of the Cas2-3 fusion's division of labour unresolved.
What would resolve it: Run the standard Cas3 biochemical panel (single-stranded and double-stranded DNA nuclease assays, directional unwinding, ATPase) on purified PA14 Cas2-3 and on isolated HD and helicase domains, then annotate the specific children of GO:0004536 and GO:0004386 that the results support.
Provenance (the field's own admissions):
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