Q46896 · CAS1_ECOLI

Function

function

CRISPR (clustered regularly interspaced short palindromic repeat), is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids) (PubMed:21255106, PubMed:24793649, PubMed:24920831).
CRISPR clusters contain sequences complementary to antecedent mobile elements and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). The Cas1-Cas2 complex is involved in CRISPR adaptation, the first stage of CRISPR immunity, being required for the addition/removal of CRISPR spacers at the leader end of the CRISPR locus (PubMed:24793649, PubMed:24920831, PubMed:25707795).
The Cas1-Cas2 complex introduces staggered nicks into both strands of the CRISPR array near the leader repeat and joins the 5'-ends of the repeat strands with the 3'-ends of the new spacer sequence (PubMed:24920831).
Spacer DNA integration requires supercoiled target DNA and 3'-OH ends on the inserted (spacer) DNA and probably initiates with a nucleophilic attack of the C 3'-OH end of the protospacer on the minus strand of the first repeat sequence (PubMed:25707795).
Expression of Cas1-Cas2 in a strain lacking both genes permits spacer acquisition (PubMed:24793649, PubMed:24920831).
Non-specifically binds DNA; the Cas1-Cas2 complex preferentially binds CRISPR-locus DNA (PubMed:24793649).
Highest binding is seen to a dual forked DNA complex with 3'-overhangs and a protospacer-adjacent motif-complement specifically positioned (PubMed:26478180).
The protospacer DNA lies across a flat surface extending from 1 Cas1 dimer, across the Cas2 dimer and contacting the other Cas1 dimer; the 23 bp-long ds section of the DNA is bracketed by 1 Tyr-22 from each of the Cas1 dimers (PubMed:26478180, PubMed:26503043).
Cas1 cuts within the 3'-overhang, to generate a 33-nucleotide DNA that is probably incorporated into the CRISPR leader by a cut-and-paste mechanism (PubMed:26478180).
Cas1 alone endonucleolytically cleaves linear ssRNA, ssDNA and short (34 base) dsDNA as well as branched DNA substrates such as Holliday junctions, replication forks and 5'-flap DNA substrates (PubMed:21219465).
In vitro catalyzes a concerted transesterification reaction on branched DNA, as would be expected during integration of protospacers into the CRISPR leader sequence; Cas2 is not required in vitro. This reaction requires a 3'-OH group at the branch point (PubMed:26284603).
Genetic interactions suggest Cas1 interacts with components of the RecBC and RuvB DNA repair systems (PubMed:21219465).

Cofactor

Mg2+ (UniProtKB | Rhea| CHEBI:18420 )

Note: Protospacer integration in vitro also occurs with Mn2+ and also requires low concentrations of KCl (PubMed:25707795).
The transesterification function works equally well with Mg2+, Mn2+ or Co2+ in vitro (PubMed:26284603).

Activity regulation

Nuclease activity partially inhibited by CasE (PubMed:21219465).

Features

Showing features for binding site.

TypeIDPosition(s)Description
Binding site141Mg2+ (UniProtKB | ChEBI)
Binding site208Mg2+ (UniProtKB | ChEBI)
Binding site221Mg2+ (UniProtKB | ChEBI)

GO annotations

AspectTerm
Cellular Componentcytoplasm
Molecular Function5'-flap endonuclease activity
Molecular Functioncrossover junction DNA endonuclease activity
Molecular FunctionDNA binding
Molecular Functionidentical protein binding
Molecular Functionmetal ion binding
Molecular Functionprotein homodimerization activity
Biological ProcessCRISPR-cas system
Biological Processdefense response to virus
Biological ProcessDNA damage response
Biological ProcessDNA repair
Biological Processmaintenance of CRISPR repeat elements

Keywords

Enzyme and pathway databases

Names & Taxonomy

Protein names

  • Recommended name
    CRISPR-associated endonuclease Cas1
  • EC number

Gene names

    • Name
      ygbT
    • Synonyms
      cas1
    • Ordered locus names
      b2755, JW2725

Organism names

  • Taxonomic identifier
  • Strains
    • K12 / MG1655 / ATCC 47076
    • K12 / W3110 / ATCC 27325 / DSM 5911
  • Taxonomic lineage
    Bacteria > Pseudomonadota > Gammaproteobacteria > Enterobacterales > Enterobacteriaceae > Escherichia

Accessions

  • Primary accession
    Q46896
  • Secondary accessions
    • Q2MA74

Proteomes

Subcellular Location

Cytoplasm
Note: In 15% of cell localizes to discrete nucleoid foci (probable DNA damage sites) upon treatment with mitomycin C (MMC) for 2 hours (PubMed:21219465).

Keywords

Phenotypes & Variants

Disruption phenotype

Not essential. Increased sensitivity to MMC and UV light; double ygbT-ruvA, ruvB or ruvC disruptions have no further phenotype suggesting Cas1 functions in the same DNA repair pathway (PubMed:21219465).
Function in DNA repair also seems to require CRISPRs (PubMed:21219465).
Cells elongate after 2 hours growth in MMC; they are even longer in double ygbT-ruvA, ruvB or ruvC disruptions, suggesting Cas1 may also function in chromosome segregation (PubMed:21219465).
Loss of plasmid silencing (PubMed:21255106).

Features

Showing features for mutagenesis.

TypeIDPosition(s)Description
Mutagenesis22Slightly decreased spacer acquisition in vivo.
Mutagenesis22Nearly wild-type spacer acquisition in vivo.
Mutagenesis41Dramatically decreased spacer acquisition in vivo.
Mutagenesis59Loss of spacer acquisition in vivo, decreased protospacer binding.
Mutagenesis59Dramatically decreased spacer acquisition in vitro, 250-fold decreased affinity for protospacer DNA.
Mutagenesis66Dramatically decreased spacer acquisition in vitro, 250-fold decreased affinity for protospacer DNA.
Mutagenesis66Dramatically decreased spacer acquisition in vivo.
Mutagenesis84Decreased spacer acquisition in vivo.
Mutagenesis84Dramatically decreased spacer acquisition in vivo.
Mutagenesis141No cleavage of any substrates, no restoration of UV or mitomycin C (MMC) resistance (PubMed:21219465).
Loss of spacer acquisition in vivo (PubMed:24793649).
Mutagenesis149No effect on in vitro protospacer integration.
Mutagenesis165No effect on in vitro protospacer integration (PubMed:25707795).
Alone significantly decreased protospacer acquisition in vivo (PubMed:26478180).
Loss of protospacer acquisition, decreased protospacer binding; in association with A-170, significantly decreased protospacer binding; in association with A-217 (PubMed:26478180).
Mutagenesis170Alone significantly decreased protospacer acquisition in vivo (PubMed:26478180).
Decreased protospacer binding; in association with A-170 (PubMed:26478180).
Mutagenesis184No cleavage of any substrates.
Mutagenesis188Partial inhibition of cleavage (PubMed:21219465).
No effect on in vitro protospacer integration (PubMed:25707795).
Significantly decreased protospacer acquisition in vivo (PubMed:26478180).
Mutagenesis208No cleavage of any substrates, no restoration of UV or MMC resistance (PubMed:21219465).
Loss of spacer acquisition in vivo (PubMed:24793649, PubMed:25707795, PubMed:26478180).
Mutagenesis211No cleavage of any substrates.
Mutagenesis217No effect on in vitro protospacer integration (PubMed:25707795).
Alone significantly decreased protospacer acquisition in vivo (PubMed:26478180).
Significantly decreased protospacer binding; in association with A-165 (PubMed:26478180).
Mutagenesis218No cleavage of any substrates, no restoration of UV or MMC resistance (PubMed:21219465).
Loss of spacer acquisition in vivo (PubMed:24793649).
Mutagenesis221No cleavage of any substrates (PubMed:21219465).
Loss of spacer acquisition in vivo (PubMed:24793649, PubMed:24920831, PubMed:25707795).
No cleavage of CRISPR leader in preparation for spacer integration (PubMed:24920831).
Mutagenesis224No cleavage of any substrates (PubMed:21219465).
Loss of spacer acquisition in vivo (PubMed:24793649, PubMed:25707795).
Mutagenesis245No effect on spacer acquisition.
Mutagenesis245Decreased spacer acquisition.
Mutagenesis245Dramatically decreased spacer acquisition in vivo.
Mutagenesis245-248Loss of spacer acquisition in vivo.
Mutagenesis248Dramatically decreased spacer acquisition in vivo.
Mutagenesis252No effect on spacer acquisition.
Mutagenesis252Loss of spacer acquisition, no Cas1-Cas2 complex formation, loss of CRISPRDNA-binding by complex. Protein is stable and dimerizes.
Mutagenesis256No effect on spacer acquisition.
Mutagenesis256Loss of spacer acquisition.
Mutagenesis256-259Loss of spacer acquisition in vivo.
Mutagenesis282-305No effect on spacer acquisition, Cas1-Cas2 complex formation or CRISPRDNA-binding by complex.
Mutagenesis291No effect on spacer acquisition.

PTM/Processing

Features

Showing features for chain.

TypeIDPosition(s)Description
ChainPRO_00001693151-305CRISPR-associated endonuclease Cas1

Proteomic databases

Expression

Induction

Repressed by H-NS (PubMed:20132443).
Activated by LeuO (PubMed:19429622).
Activated by the BaeSR two-component regulatory system, possibly due to envelope stress (PubMed:21255106).
Part of the casABCDE-ygbT-ygbF operon (PubMed:19429622).

Interaction

Subunit

Homodimer (PubMed:21219465).
Part of the Cas1-Cas2 complex (PubMed:24793649, PubMed:24920831, PubMed:25707795, PubMed:26478180, PubMed:26503043, Ref.11). Interacts with RecB, RecC, RuvB, CasC and CasE (PubMed:21219465).
Forms a hexamer with 2 Cas1 dimers sandwiching a Cas2 dimer (PubMed:24793649, PubMed:26478180).
The DNA lies across a flat surface extending from 1 Cas1 dimer, across the Cas2 dimer and contacting the other Cas1 dimer. Only 1 Cas1 protein from each dimer is catalytic, the other interacts with the Cas2 dimer and possibly target DNA (PubMed:26478180, PubMed:26503043).

Binary interactions

TypeEntry 1Entry 2Number of experimentsIntact
BINARY Q46896hprS P763393EBI-1130209, EBI-554869
BINARY Q46896ygbF P459568EBI-1130209, EBI-9150552
BINARY Q46896ygbT Q468964EBI-1130209, EBI-1130209

Protein-protein interaction databases

Family & Domains

Features

Showing features for region.

TypeIDPosition(s)Description
Region96-278Sufficient for cleavage of ssRNA, ssDNA and Holliday junction DNA
Region278-305Disordered

Domain

Substrate DNA-binding induces large structural changes that generate a surface for DNA-binding across the Cas2 dimer and formation of an optimal catalytic site (PubMed:26478180).

Sequence similarities

Phylogenomic databases

Family and domain databases

Sequence

  • Sequence status
    Complete
  • Length
    305
  • Mass (Da)
    33,194
  • Last updated
    1996-11-01 v1
  • Checksum
    01A31BA98453D8A5
MTWLPLNPIPLKDRVSMIFLQYGQIDVIDGAFVLIDKTGIRTHIPVGSVACIMLEPGTRVSHAAVRLAAQVGTLLVWVGEAGVRVYASGQPGGARSDKLLYQAKLALDEDLRLKVVRKMFELRFGEPAPARRSVEQLRGIEGSRVRATYALLAKQYGVTWNGRRYDPKDWEKGDTINQCISAATSCLYGVTEAAILAAGYAPAIGFVHTGKPLSFVYDIADIIKFDTVVPKAFEIARRNPGEPDREVRLACRDIFRSSKTLAKLIPLIEDVLAAGEIQPPAPPEDAQPVAIPLPVSLGDAGHRSS

Keywords

Sequence databases

Nucleotide SequenceProtein SequenceMolecule TypeStatus
U29579
EMBL· GenBank· DDBJ
AAA69265.1
EMBL· GenBank· DDBJ
Genomic DNA
U00096
EMBL· GenBank· DDBJ
AAC75797.1
EMBL· GenBank· DDBJ
Genomic DNA
AP009048
EMBL· GenBank· DDBJ
BAE76832.1
EMBL· GenBank· DDBJ
Genomic DNA

Genome annotation databases

Similar Proteins

Disclaimer

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