CAS2_ECOLI
ID CAS2_ECOLI Reviewed; 94 AA.
AC P45956; Q2MA75;
DT 01-NOV-1995, integrated into UniProtKB/Swiss-Prot.
DT 30-MAY-2000, sequence version 2.
DT 03-AUG-2022, entry version 131.
DE RecName: Full=CRISPR-associated endoribonuclease Cas2;
DE EC=3.1.-.-;
GN Name=ygbF; Synonyms=cas2; OrderedLocusNames=b2754, JW5438;
OS Escherichia coli (strain K12).
OC Bacteria; Proteobacteria; Gammaproteobacteria; Enterobacterales;
OC Enterobacteriaceae; Escherichia.
OX NCBI_TaxID=83333;
RN [1]
RP NUCLEOTIDE SEQUENCE [GENOMIC DNA].
RC STRAIN=K12;
RX PubMed=2656660; DOI=10.1128/jb.171.6.3553-3556.1989;
RA Nakata A., Amemura M., Makino K.;
RT "Unusual nucleotide arrangement with repeated sequences in the Escherichia
RT coli K-12 chromosome.";
RL J. Bacteriol. 171:3553-3556(1989).
RN [2]
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=K12 / MG1655 / ATCC 47076;
RX PubMed=9278503; DOI=10.1126/science.277.5331.1453;
RA Blattner F.R., Plunkett G. III, Bloch C.A., Perna N.T., Burland V.,
RA Riley M., Collado-Vides J., Glasner J.D., Rode C.K., Mayhew G.F.,
RA Gregor J., Davis N.W., Kirkpatrick H.A., Goeden M.A., Rose D.J., Mau B.,
RA Shao Y.;
RT "The complete genome sequence of Escherichia coli K-12.";
RL Science 277:1453-1462(1997).
RN [3]
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=K12 / W3110 / ATCC 27325 / DSM 5911;
RX PubMed=16738553; DOI=10.1038/msb4100049;
RA Hayashi K., Morooka N., Yamamoto Y., Fujita K., Isono K., Choi S.,
RA Ohtsubo E., Baba T., Wanner B.L., Mori H., Horiuchi T.;
RT "Highly accurate genome sequences of Escherichia coli K-12 strains MG1655
RT and W3110.";
RL Mol. Syst. Biol. 2:E1-E5(2006).
RN [4]
RP IDENTIFICATION.
RX PubMed=7567469; DOI=10.1093/nar/23.17.3554;
RA Borodovsky M., McIninch J., Koonin E.V., Rudd K.E., Medigue C., Danchin A.;
RT "Detection of new genes in a bacterial genome using Markov models for three
RT gene classes.";
RL Nucleic Acids Res. 23:3554-3562(1995).
RN [5]
RP OPERON STRUCTURE, AND INDUCTION BY LEUO.
RC STRAIN=K12 / BW25113;
RX PubMed=19429622; DOI=10.1128/jb.00108-09;
RA Shimada T., Yamamoto K., Ishihama A.;
RT "Involvement of the leucine response transcription factor LeuO in
RT regulation of the genes for sulfa drug efflux.";
RL J. Bacteriol. 191:4562-4571(2009).
RN [6]
RP REPRESSION BY H-NS.
RC STRAIN=K12;
RX PubMed=20132443; DOI=10.1111/j.1365-2958.2010.07073.x;
RA Pul U., Wurm R., Arslan Z., Geissen R., Hofmann N., Wagner R.;
RT "Identification and characterization of E. coli CRISPR-cas promoters and
RT their silencing by H-NS.";
RL Mol. Microbiol. 75:1495-1512(2010).
RN [7]
RP INDUCTION BY BAER, ROLE IN PLASMID SILENCING, AND DISRUPTION PHENOTYPE.
RC STRAIN=K12 / BW25113;
RX PubMed=21255106; DOI=10.1111/j.1365-2958.2010.07482.x;
RA Perez-Rodriguez R., Haitjema C., Huang Q., Nam K.H., Bernardis S., Ke A.,
RA DeLisa M.P.;
RT "Envelope stress is a trigger of CRISPR RNA-mediated DNA silencing in
RT Escherichia coli.";
RL Mol. Microbiol. 79:584-599(2011).
RN [8]
RP FUNCTION AS A SPACER INTEGRASE, AND SUBUNIT.
RC STRAIN=K12 / MG1655 / ATCC 47076;
RX PubMed=24920831; DOI=10.1093/nar/gku510;
RA Arslan Z., Hermanns V., Wurm R., Wagner R., Pul U.;
RT "Detection and characterization of spacer integration intermediates in type
RT I-E CRISPR-Cas system.";
RL Nucleic Acids Res. 42:7884-7893(2014).
RN [9]
RP FUNCTION, SUBUNIT, AND MUTAGENESIS OF GLU-9 AND 79-THR--VAL-94.
RC STRAIN=K12 / MG1655 / ATCC 47076;
RX PubMed=25707795; DOI=10.1038/nature14237;
RA Nunez J.K., Lee A.S., Engelman A., Doudna J.A.;
RT "Integrase-mediated spacer acquisition during CRISPR-Cas adaptive
RT immunity.";
RL Nature 519:193-198(2015).
RN [10]
RP X-RAY CRYSTALLOGRAPHY (1.1 ANGSTROMS).
RA Nocek B., Skarina T., Brown G., Yakunin A., Joachimiak A.;
RT "Crystal structure of a putative ssRNA endonuclease Cas2, CRISPR adaptation
RT protein from E.coli.";
RL Submitted (AUG-2013) to the PDB data bank.
RN [11]
RP X-RAY CRYSTALLOGRAPHY (2.3 ANGSTROMS) IN COMPLEX WITH CAS1, FUNCTION IN
RP SPACER ACQUISITION, INTERACTION WITH CAS1 (YGBT), SUBUNIT, AND MUTAGENESIS
RP OF GLU-9; ASN-10; ARG-14; ARG-16; ARG-18; ARG-27; GLU-65; 79-THR--VAL-94
RP AND ASP-84.
RC STRAIN=K12 / MG1655 / ATCC 47076;
RX PubMed=24793649; DOI=10.1038/nsmb.2820;
RA Nunez J.K., Kranzusch P.J., Noeske J., Wright A.V., Davies C.W.,
RA Doudna J.A.;
RT "Cas1-Cas2 complex formation mediates spacer acquisition during CRISPR-Cas
RT adaptive immunity.";
RL Nat. Struct. Mol. Biol. 21:528-534(2014).
RN [12]
RP X-RAY CRYSTALLOGRAPHY (2.70 ANGSTROMS) IN COMPLEX WITH CAS1, AND SUBUNIT.
RA Tamulaitiene G., Sinkunas T., Silanskas A., Gasiunas G., Grazulis S.,
RA Siksnys V.;
RT "Crystal structure of E.coli Cas1-Cas2 complex.";
RL Submitted (MAY-2014) to the PDB data bank.
RN [13]
RP X-RAY CRYSTALLOGRAPHY (2.60 ANGSTROMS) OF 1-78 IN COMPLEX WITH CAS1 AND
RP DNA, FUNCTION, SUBUNIT, DOMAIN, DNA-BINDING, AND MUTAGENESIS OF
RP 14-ARG--ARG-16; 38-LYS--ARG-40 AND 77-ARG-ARG-78.
RC STRAIN=K12;
RX PubMed=26478180; DOI=10.1016/j.cell.2015.10.008;
RA Wang J., Li J., Zhao H., Sheng G., Wang M., Yin M., Wang Y.;
RT "Structural and mechanistic basis of PAM-dependent spacer acquisition in
RT CRISPR-Cas systems.";
RL Cell 163:840-853(2015).
RN [14]
RP X-RAY CRYSTALLOGRAPHY (2.95 ANGSTROMS) IN COMPLEX WITH CAS1 AND DNA,
RP FUNCTION, SUBUNIT, DNA-BINDING, AND MUTAGENESIS OF ARG-16; ARG-77 AND
RP ARG-78.
RX PubMed=26503043; DOI=10.1038/nature15760;
RA Nunez J.K., Harrington L.B., Kranzusch P.J., Engelman A.N., Doudna J.A.;
RT "Foreign DNA capture during CRISPR-Cas adaptive immunity.";
RL Nature 527:535-538(2015).
CC -!- FUNCTION: CRISPR (clustered regularly interspaced short palindromic
CC repeat), is an adaptive immune system that provides protection against
CC mobile genetic elements (viruses, transposable elements and conjugative
CC plasmids) (PubMed:21255106, PubMed:24920831, PubMed:24793649). CRISPR
CC clusters contain sequences complementary to antecedent mobile elements
CC and target invading nucleic acids. CRISPR clusters are transcribed and
CC processed into CRISPR RNA (crRNA). The Cas1-Cas2 complex is involved in
CC CRISPR adaptation, the first stage of CRISPR immunity, being required
CC for the addition/removal of CRISPR spacers at the leader end of the
CC CRISPR locus (PubMed:24920831, PubMed:25707795, PubMed:24793649). The
CC Cas1-Cas2 complex introduces staggered nicks into both strands of the
CC CRISPR array near the leader repeat and joins the 5'-ends of the repeat
CC strands with the 3'-ends of the new spacer sequence (PubMed:24920831).
CC Spacer DNA integration requires supercoiled target DNA and 3'-OH ends
CC on the inserted (spacer) DNA and probably initiates with a nucleophilic
CC attack of the C 3'-OH end of the protospacer on the minus strand of the
CC first repeat sequence (PubMed:25707795). Expression of Cas1-Cas2 in a
CC strain lacking both genes permits spacer acquisition (PubMed:24793649,
CC PubMed:24920831). Cas2 not seen to bind DNA alone; the Cas1-Cas2
CC complex preferentially binds CRISPR-locus DNA (PubMed:24793649).
CC Highest binding is seen to a dual forked DNA complex with 3'-overhangs
CC and a protospacer-adjacent motif-complement specifically positioned
CC (PubMed:26478180). The protospacer DNA lies across a flat surface
CC extending from 1 Cas1 dimer, across the Cas2 dimer and contacting the
CC other Cas1 dimer; the 23 bp-long ds section of the DNA is bracketed by
CC 1 Tyr-22 from each of the Cas1 dimers (PubMed:26478180,
CC PubMed:26503043). Cas1 cuts within the 3'-overhang, to generate a 33-
CC nucleotide DNA that is probably incorporated into the CRISPR leader by
CC a cut-and-paste mechanism (PubMed:26478180). This subunit's probable
CC nuclease activity is not required for spacer acquisition
CC (PubMed:24793649). {ECO:0000269|PubMed:21255106,
CC ECO:0000269|PubMed:24793649, ECO:0000269|PubMed:24920831,
CC ECO:0000269|PubMed:26478180}.
CC -!- SUBUNIT: Homodimer (Ref.10). Part of the Cas1-Cas2 complex
CC (PubMed:24920831, PubMed:24793649, PubMed:25707795, Ref.12,
CC PubMed:26478180, PubMed:26503043). Forms a hexamer with 2 Cas1 dimers
CC sandwiching a Cas2 dimer (PubMed:24793649). The DNA lies across a flat
CC surface extending from 1 Cas1 dimer, across the Cas2 dimer and
CC contacting the other Cas1 dimer. Only 1 Cas1 protein from each dimer is
CC catalytic, the other interacts with the Cas2 dimer and possibly target
CC DNA (PubMed:26478180, PubMed:26503043). {ECO:0000269|PubMed:24793649,
CC ECO:0000269|PubMed:24920831, ECO:0000269|PubMed:25707795,
CC ECO:0000269|PubMed:26478180, ECO:0000269|PubMed:26503043,
CC ECO:0000305|Ref.10, ECO:0000305|Ref.12}.
CC -!- INTERACTION:
CC P45956; Q46896: ygbT; NbExp=8; IntAct=EBI-9150552, EBI-1130209;
CC -!- INDUCTION: Repressed by H-NS (PubMed:20132443). Activated by LeuO
CC (PubMed:19429622). Activated by the BaeSR two-component regulatory
CC system, possibly due to envelope stress (PubMed:21255106). Part of the
CC casABCDE-ygbT-ygbF operon (PubMed:19429622).
CC {ECO:0000269|PubMed:19429622, ECO:0000269|PubMed:20132443,
CC ECO:0000269|PubMed:21255106}.
CC -!- DOMAIN: Substrate DNA-binding induces large structural changes that
CC generate a surface for DNA-binding across the Cas2 dimer and formation
CC of an optimal catalytic site (PubMed:26478180).
CC {ECO:0000269|PubMed:26478180}.
CC -!- DISRUPTION PHENOTYPE: Loss of plasmid silencing (PubMed:21255106).
CC {ECO:0000269|PubMed:21255106}.
CC -!- SIMILARITY: Belongs to the CRISPR-associated endoribonuclease Cas2
CC protein family. E.coli-subtype subfamily. {ECO:0000305}.
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DR EMBL; M27059; -; NOT_ANNOTATED_CDS; Unassigned_DNA.
DR EMBL; U29579; AAA69264.1; -; Genomic_DNA.
DR EMBL; U00096; AAC75796.2; -; Genomic_DNA.
DR EMBL; AP009048; BAE76831.1; -; Genomic_DNA.
DR PIR; F65056; F65056.
DR RefSeq; NP_417234.2; NC_000913.3.
DR RefSeq; WP_001381369.1; NZ_LN832404.1.
DR PDB; 4MAK; X-ray; 1.10 A; A/B=1-94.
DR PDB; 4P6I; X-ray; 2.30 A; A/B=1-94.
DR PDB; 4QDL; X-ray; 2.70 A; E/F=1-94.
DR PDB; 5DLJ; X-ray; 2.60 A; E/F=1-78.
DR PDB; 5DQT; X-ray; 3.10 A; E/F/M/N=1-94.
DR PDB; 5DQU; X-ray; 4.50 A; E/F=1-94.
DR PDB; 5DQZ; X-ray; 2.70 A; E/F=1-94.
DR PDB; 5DS4; X-ray; 3.20 A; E/F=1-94.
DR PDB; 5DS5; X-ray; 2.95 A; E/F=1-94.
DR PDB; 5DS6; X-ray; 3.35 A; E/F=1-94.
DR PDB; 5VVJ; X-ray; 3.89 A; E/F=1-94.
DR PDB; 5VVK; X-ray; 2.90 A; E/F=1-94.
DR PDB; 5VVL; X-ray; 3.31 A; E/F=1-94.
DR PDB; 5WFE; EM; 3.64 A; E/F=1-94.
DR PDBsum; 4MAK; -.
DR PDBsum; 4P6I; -.
DR PDBsum; 4QDL; -.
DR PDBsum; 5DLJ; -.
DR PDBsum; 5DQT; -.
DR PDBsum; 5DQU; -.
DR PDBsum; 5DQZ; -.
DR PDBsum; 5DS4; -.
DR PDBsum; 5DS5; -.
DR PDBsum; 5DS6; -.
DR PDBsum; 5VVJ; -.
DR PDBsum; 5VVK; -.
DR PDBsum; 5VVL; -.
DR PDBsum; 5WFE; -.
DR AlphaFoldDB; P45956; -.
DR SMR; P45956; -.
DR BioGRID; 4259585; 139.
DR BioGRID; 851545; 2.
DR ComplexPortal; CPX-996; Cas1-Cas2 complex.
DR DIP; DIP-12109N; -.
DR IntAct; P45956; 3.
DR STRING; 511145.b2754; -.
DR PaxDb; P45956; -.
DR PRIDE; P45956; -.
DR EnsemblBacteria; AAC75796; AAC75796; b2754.
DR EnsemblBacteria; BAE76831; BAE76831; BAE76831.
DR GeneID; 947213; -.
DR KEGG; ecj:JW5438; -.
DR KEGG; eco:b2754; -.
DR PATRIC; fig|1411691.4.peg.3984; -.
DR EchoBASE; EB2694; -.
DR eggNOG; COG0847; Bacteria.
DR HOGENOM; CLU_151313_1_0_6; -.
DR InParanoid; P45956; -.
DR OMA; TNNEQGF; -.
DR PhylomeDB; P45956; -.
DR BioCyc; EcoCyc:EG12845-MON; -.
DR PRO; PR:P45956; -.
DR Proteomes; UP000000318; Chromosome.
DR Proteomes; UP000000625; Chromosome.
DR GO; GO:0003677; F:DNA binding; IEA:UniProtKB-KW.
DR GO; GO:0004519; F:endonuclease activity; IEA:UniProtKB-KW.
DR GO; GO:0099048; P:CRISPR-cas system; IDA:ComplexPortal.
DR GO; GO:0051607; P:defense response to virus; IEA:UniProtKB-KW.
DR GO; GO:0043571; P:maintenance of CRISPR repeat elements; IDA:ComplexPortal.
DR InterPro; IPR010152; CRISPR-assoc_prot_Cas2_sub.
DR Pfam; PF09707; Cas_Cas2CT1978; 1.
DR TIGRFAMs; TIGR01873; cas_CT1978; 1.
PE 1: Evidence at protein level;
KW 3D-structure; Antiviral defense; DNA-binding; Endonuclease; Hydrolase;
KW Nuclease; Reference proteome.
FT CHAIN 1..94
FT /note="CRISPR-associated endoribonuclease Cas2"
FT /id="PRO_0000169312"
FT MUTAGEN 9
FT /note="E->A,R: No effect on spacer acquisition, Cas1-Cas2
FT complex formation or CRISPR DNA-binding by complex."
FT /evidence="ECO:0000269|PubMed:24793649,
FT ECO:0000269|PubMed:25707795"
FT MUTAGEN 10
FT /note="N->A: No effect on spacer acquisition."
FT /evidence="ECO:0000269|PubMed:24793649"
FT MUTAGEN 14..16
FT /note="RLR->ALA: No in vivspacer acquisition, significantly
FT decreased protospacer binding."
FT /evidence="ECO:0000269|PubMed:26478180"
FT MUTAGEN 14
FT /note="R->A: Slight decrease in spacer acquisition."
FT /evidence="ECO:0000269|PubMed:24793649"
FT MUTAGEN 16
FT /note="R->A: Slight decrease in spacer acquisition."
FT /evidence="ECO:0000269|PubMed:24793649"
FT MUTAGEN 16
FT /note="R->E: Dramatically decreased spacer acquisition in
FT vivo."
FT /evidence="ECO:0000269|PubMed:26503043"
FT MUTAGEN 18
FT /note="R->A: Very little spacer acquisition."
FT /evidence="ECO:0000269|PubMed:24793649"
FT MUTAGEN 27
FT /note="R->A: Slight decrease in spacer acquisition."
FT /evidence="ECO:0000269|PubMed:24793649"
FT MUTAGEN 38..40
FT /note="KIR->AIA: Very little in vivo spacer acquisition."
FT /evidence="ECO:0000269|PubMed:26478180"
FT MUTAGEN 65
FT /note="E->A: No effect on spacer acquisition."
FT /evidence="ECO:0000269|PubMed:24793649"
FT MUTAGEN 65
FT /note="E->R: Slight decrease in spacer acquisition, Cas1-
FT Cas2 complex formation or CRISPR DNA-binding by complex.
FT Loss of spacer acquisition; when associated with R-84."
FT /evidence="ECO:0000269|PubMed:24793649"
FT MUTAGEN 77..78
FT /note="RR->AA: No spacer acquisition, significantly
FT decreased protospacer binding."
FT /evidence="ECO:0000269|PubMed:26478180"
FT MUTAGEN 77
FT /note="R->E: No change in spacer acquisition in vivo."
FT /evidence="ECO:0000269|PubMed:26503043"
FT MUTAGEN 78
FT /note="R->E: Dramatically decreased spacer acquisition in
FT vivo."
FT /evidence="ECO:0000269|PubMed:26503043"
FT MUTAGEN 79..94
FT /note="Missing: Loss of spacer acquisition, no Cas1-Cas2
FT complex formation, loss of CRISPR DNA-binding by complex
FT (beta6-beta7 deletion)."
FT /evidence="ECO:0000269|PubMed:24793649,
FT ECO:0000269|PubMed:25707795"
FT MUTAGEN 84
FT /note="D->A: No effect on spacer acquisition."
FT /evidence="ECO:0000269|PubMed:24793649"
FT MUTAGEN 84
FT /note="D->R: Slight decrease in spacer acquisition. Loss of
FT spacer acquisition; when associated with R-65."
FT /evidence="ECO:0000269|PubMed:24793649"
FT STRAND 3..10
FT /evidence="ECO:0007829|PDB:4MAK"
FT HELIX 13..22
FT /evidence="ECO:0007829|PDB:4MAK"
FT STRAND 23..27
FT /evidence="ECO:0007829|PDB:4MAK"
FT STRAND 30..34
FT /evidence="ECO:0007829|PDB:4MAK"
FT HELIX 37..50
FT /evidence="ECO:0007829|PDB:4MAK"
FT STRAND 55..61
FT /evidence="ECO:0007829|PDB:4MAK"
FT STRAND 63..66
FT /evidence="ECO:0007829|PDB:4MAK"
FT STRAND 68..73
FT /evidence="ECO:0007829|PDB:4MAK"
FT STRAND 79..83
FT /evidence="ECO:0007829|PDB:4P6I"
FT STRAND 86..91
FT /evidence="ECO:0007829|PDB:4P6I"
SQ SEQUENCE 94 AA; 10518 MW; D1C159D924B477B4 CRC64;
MSMLVVVTEN VPPRLRGRLA IWLLEVRAGV YVGDVSAKIR EMIWEQIAGL AEEGNVVMAW
ATNTETGFEF QTFGLNRRTP VDLDGLRLVS FLPV
