Cas1 is the protospacer integrase of the Pseudomonas aeruginosa UCBPP-PA14 type I-F (Yersinia-subtype) CRISPR-Cas system and the only protein component conserved across all CRISPR system subtypes. It is a homodimeric, metal-dependent, DNA-specific endonuclease: Mn(2+) supports cleavage of both single- and double-stranded DNA while Mg(2+) supports only double-stranded substrates, and the divalent metal site formed by Glu190, His254 and His268 is required for activity. In the adaptation stage of the pathway Cas1 captures a fragment of invading phage or plasmid DNA and integrates it as a new spacer at the leader-proximal end of a CRISPR array, which is what makes the immunity heritable; mutation of the metal-coordinating Asp268 abolishes acquisition. Type I-F systems encode no standalone Cas2, so the non-catalytic partner that measures the protospacer duplex is instead the N-terminal Cas2 module of the fused Cas2-3 protein. Cas1 is dispensable for the interference stage, in which the Csy surveillance complex and Cas3 destroy the target, but the type I-F system nevertheless requires an intact Csy complex and crRNA for acquisition, coupling the two stages more tightly than in type I-E.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003676 nucleic acid binding | IEA GO_REF:0000002 | MODIFY | Summary: Correct but needlessly general. The nucleic acid this protein acts on was directly determined to be DNA, and UniProt carries the DNA-binding keyword for Q02ML7 on that basis. GO:0003677 DNA binding keeps the specificity that was actually measured. Reason: Substrate specificity is experimentally established for this protein, so the generic parent discards information rather than being appropriately cautious. Proposed replacements: DNA binding Supporting Evidence: PMID:19523907 the Cas1 protein is a metal-dependent DNA-specific endonuclease that produces double-stranded DNA fragments of approximately 80 base pairs in length |
| GO:0004519 endonuclease activity | IEA GO_REF:0000120 | MODIFY | Summary: The parent of the term already annotated from a second InterPro signature. The activity measured on this protein is DNA-specific, so GO:0004520 DNA endonuclease activity is the supported child and this row should consolidate onto it rather than stand as a separate, vaguer claim. Reason: Consolidation, not addition: GO:0004520 is already asserted for this protein from InterPro IPR019857 and is directly supported by assay. Proposed replacements: DNA endonuclease activity Supporting Evidence: PMID:19523907 the Cas1 protein is a metal-dependent DNA-specific endonuclease that produces double-stranded DNA fragments of approximately 80 base pairs in length |
| GO:0004520 DNA endonuclease activity | IEA GO_REF:0000002 | ACCEPT | Summary: Core molecular function. Although the evidence code is electronic, the activity was measured directly on this protein: it degrades single- and double-stranded DNA to roughly 80 bp products, and activity is lost on mutation of the metal site or chelation of metal ions. Supporting Evidence: PMID:19523907 the Cas1 protein is a metal-dependent DNA-specific endonuclease that produces double-stranded DNA fragments of approximately 80 base pairs in length PMID:19523907 Mutation of metal ion-binding residues, chelation of metal ions, or metal-ion substitution inhibits Cas1-catalyzed DNA degradation |
| GO:0042802 identical protein binding | IPI PMID:19523907 Structural basis for DNase activity of a conserved protein i... | MODIFY | Summary: The IntAct self-interaction records a genuine homodimer, which UniProt states for this protein from the same study. GO:0042803 protein homodimerization activity says that directly, and is the term the Escherichia coli orthologue Cas1 (UniProtKB:Q46896) carries by IDA, so the more informative child is the family convention. Reason: Identical protein binding understates a defined quaternary structure; the orthologue comparator carries the homodimerization term. Proposed replacements: protein homodimerization activity Supporting Evidence: PMID:19523907 The 2.2 A crystal structure of the Cas1 protein reveals a distinct fold and a conserved divalent metal ion-binding site |
| GO:0043571 maintenance of CRISPR repeat elements | IEA GO_REF:0000120 | ACCEPT | Summary: Correct. The term's definition names capture of new spacer elements, which is the step Cas1 catalyses, and the signatures cited are the Cas1 family and its Yersinia-subtype subfamily. Supporting Evidence: PMID:19523907 cas1) encodes the only universally conserved protein component of CRISPR immune systems |
| GO:0043571 maintenance of CRISPR repeat elements | IMP PMID:26586803 Foreign DNA acquisition by the I-F CRISPR-Cas system require... | ACCEPT | Summary: Core biological process, with gene-specific experimental grounding: a Cas1 D268A substitution at a metal-coordinating residue abolished new spacer acquisition by the Pseudomonas aeruginosa type I-F system. The system was studied transplanted into an Escherichia coli host, but the alleles tested were the Pseudomonas genes, so the annotation is correctly attributed. Supporting Evidence: PMID:26586803 This is an expected result since D268 is a conserved metal coordinating residue and substitution of the corresponding residue in E. coli Cas1 also abolishes adaptation PMID:26586803 both modes require, in addition to Cas1 and Cas2, intact Csy complex, an ortholog of the E. coli Cascade, and crRNA |
| GO:0046872 metal ion binding | IEA GO_REF:0000120 | ACCEPT | Summary: Supported for this protein beyond the electronic inference. The crystal structure resolves a divalent metal site, UniProt records Mn(2+) and Mg(2+) ligands at residues 190, 254 and 268 from PDB 3GOD, activity is inhibited by EDTA, and metal-site mutants lose catalysis. Supporting Evidence: PMID:19523907 The 2.2 A crystal structure of the Cas1 protein reveals a distinct fold and a conserved divalent metal ion-binding site PMID:19523907 Mutation of metal ion-binding residues, chelation of metal ions, or metal-ion substitution inhibits Cas1-catalyzed DNA degradation |
| GO:0051607 defense response to virus | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: Not wrong, but a step removed from what Cas1 does. Cas1 is dispensable for the interference stage that actually clears an infecting phage; its contribution to antiviral immunity is to write the spacer that a later generation's effector will use. The PA14 locus is also the one case where the antiviral phenotype has been reported both ways, and no cas1 allele has been assayed for phage resistance. Retained rather than removed, because the statement is defensible and removing a pipeline term on an argument about directness would overreach. Reason: Cas1 acts in adaptation, not interference, so it contributes to defence only indirectly; GO:0043571 and GO:0099048 carry the direct roles. Supporting Evidence: PMID:26586803 Cas1 and Cas2 are not required for CRISPR interference (22) PMID:23242138 The CRISPR-sensitive phages fail to replicate on PA14 due to the action of the CRISPR/Cas system14, but are able to replicate on PA14 |
| GO:0099048 CRISPR-cas system | IMP PMID:26586803 Foreign DNA acquisition by the I-F CRISPR-Cas system require... | NEW | Summary: Cas1 catalyses the acquisition stage that this term's definition names as the first of the system's three stages, and the D268A allele gives gene-specific evidence in this strain's system. Reason: Comparator check: the Escherichia coli orthologue Cas1 (Q46896) carries involved_in GO:0099048 by IDA from PMID:27899566 and again by IBA, as does Cas2 (P45956) and every Cascade subunit, yet no Pseudomonas aeruginosa PA14 cas gene does. The absence is a per-strain curation lag, not a convention. Checked against QuickGO: GO:0099048 is neither an ancestor nor a descendant of GO:0043571 or GO:0051607, so it is not redundant with the existing process annotations. Cas1 itself performs the integration chemistry, so this is participation rather than mere requirement. Supporting Evidence: PMID:26586803 This is an expected result since D268 is a conserved metal coordinating residue and substitution of the corresponding residue in E. coli Cas1 also abolishes adaptation PMID:19523907 cas1) encodes the only universally conserved protein component of CRISPR immune systems |
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Download this section (compressed HTML)Q: UniProt notes that this organism encodes no standalone Cas2 and that Cas1 must therefore use a different partner. Does PA14 Cas1 assemble with the Cas2 module of Cas2-3 in the same 2:1:2 arrangement as the type I-E Cas1-Cas2 integrase, and is the full-length Cas2-3 protein or a processed fragment the physiological partner?
Q: Why does type I-F acquisition require an intact Csy complex and crRNA even for naive adaptation, when type I-E naive adaptation needs only Cas1 and Cas2? Is the Csy complex supplying the substrate, or licensing Cas1 activity?
Q: The purified protein degrades DNA to roughly 80 bp fragments in vitro. Is that nuclease activity the integration chemistry seen out of context, or a separate activity, and what restrains it from damaging the host genome in vivo?
Experiment: Express the isolated Cas2 module (residues approximately 1-124 of Cas2-3) together with Cas1 in the transplanted type I-F reporter system and test whether it restores naive spacer acquisition to a cas2-3 D124 ochre strain. Pair with size-exclusion chromatography and mass photometry on the co-expressed pair to establish the stoichiometry.
Hypothesis: The Cas2 module of the fused Cas2-3 protein is the integration partner of PA14 Cas1 and is sufficient for naive acquisition when supplied separately.
Type: genetic complementation and reconstitution
Experiment: Compare acquisition in strains carrying a catalytically dead Cas3 nuclease domain, a DNA-binding-dead Cas8f K-wedge mutant, and a wild-type control, and sequence the acquired spacers to ask whether the Csy requirement maps onto target engagement or onto substrate generation.
Hypothesis: The requirement for the Csy complex in naive acquisition reflects delivery of a protospacer-length DNA intermediate rather than an allosteric effect on Cas1.
Type: genetic epistasis with spacer sequencing
What is not known — curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: The non-catalytic partner that Pseudomonas aeruginosa PA14 Cas1 works with during spacer integration has not been identified experimentally. Type I-F encodes no standalone Cas2, so the partner is presumed to be the N-terminal Cas2 module of the fused Cas2-3 protein, but no direct Cas1-Cas2-3 complex has been characterised for this organism.
OPEN BIOLOGY
What is known: The fusion itself is established, and a stop codon placed after the cas2 portion of the fused gene abolishes acquisition, so the Cas2 module is necessary. What is missing is the physical Cas1-Cas2 module interaction, its stoichiometry, and the protospacer-ruler geometry that Cas2 provides in type I-E.
Significance: The protospacer length distribution and the strand bias of primed acquisition both depend on the geometry of the integration complex, which cannot be modelled without knowing how the Cas2 module docks onto Cas1.
What would resolve it: Reconstitute PA14 Cas1 with full-length Cas2-3 and with an isolated Cas2 module, and determine the integration-complex structure.
Provenance (the field's own admissions):
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