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 29-SEP-2021, entry version 128. 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}. CC --------------------------------------------------------------------------- CC Copyrighted by the UniProt Consortium, see https://www.uniprot.org/terms CC Distributed under the Creative Commons Attribution (CC BY 4.0) License CC --------------------------------------------------------------------------- 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 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 PhylomeDB; P45956; -. DR BioCyc; EcoCyc:EG12845-MONOMER; -. 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:0051607; P:defense response to virus; IEA:UniProtKB-KW. DR GO; GO:0043571; P:maintenance of CRISPR repeat elements; IMP:EcoCyc. 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 //