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 07-SEP-2016, entry version 102. 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; Enterobacteriales; OC Enterobacteriaceae; Escherichia. OX NCBI_TaxID=83333; RN [1] RP NUCLEOTIDE SEQUENCE [GENOMIC DNA]. RC STRAIN=K12; RX PubMed=2656660; RA Nakata A., Amemura M., Makino K.; RT "Unusual nucleotide arrangement with repeated sequences in the RT Escherichia 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., RA Mau B., 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 RT MG1655 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., RA Danchin A.; RT "Detection of new genes in a bacterial genome using Markov models for RT three 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 RT and their silencing by H-NS."; RL Mol. Microbiol. 75:1495-1512(2010). RN [7] RP INDUCTION BY BAER, ROLE IN PLASMID SILENCING, AND DISRUPTION RP 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., RA Ke A., 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 RT type 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 RT adaptation 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 RP IN SPACER ACQUISITION, INTERACTION WITH CAS1 (YGBT), SUBUNIT, AND RP MUTAGENESIS OF GLU-9; ASN-10; ARG-14; ARG-16; ARG-18; ARG-27; GLU-65; RP 79-THR--VAL-94 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 RT CRISPR-Cas 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 RP 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 RP AND 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 RT in 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., RA 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 CC palindromic repeat), is an adaptive immune system that provides CC protection against mobile genetic elements (viruses, transposable CC elements and conjugative plasmids) (PubMed:21255106, CC PubMed:24920831, PubMed:24793649). CRISPR clusters contain CC sequences complementary to antecedent mobile elements and target CC invading nucleic acids. CRISPR clusters are transcribed and CC processed into CRISPR RNA (crRNA). The Cas1-Cas2 complex is CC involved in CRISPR adaptation, the first stage of CRISPR immunity, CC being required for the addition/removal of CRISPR spacers at the CC leader end of the CRISPR locus (PubMed:24920831, PubMed:25707795, CC PubMed:24793649). The Cas1-Cas2 complex introduces staggered nicks CC into both strands of the CRISPR array near the leader repeat and CC joins the 5'-ends of the repeat strands with the 3'-ends of the CC new spacer sequence (PubMed:24920831). Spacer DNA integration CC requires supercoiled target DNA and 3'-OH ends on the inserted CC (spacer) DNA and probably initiates with a nucleophilic attack of CC the C 3'-OH end of the protospacer on the minus strand of the CC first repeat sequence (PubMed:25707795). Expression of Cas1-Cas2 CC in a strain lacking both genes permits spacer acquisition CC (PubMed:24793649, PubMed:24920831). Cas2 not seen to bind DNA CC alone; the Cas1-Cas2 complex preferentially binds CRISPR-locus DNA CC (PubMed:24793649). Highest binding is seen to a dual forked DNA CC complex with 3'-overhangs and a protospacer-adjacent motif- CC complement specifically positioned (PubMed:26478180). The CC protospacer DNA lies across a flat surface extending from 1 Cas1 CC dimer, across the Cas2 dimer and contacting the other Cas1 dimer; CC the 23 bp-long ds section of the DNA is bracketed by 1 Tyr-22 from CC each of the Cas1 dimers (PubMed:26478180, PubMed:26503043). Cas1 CC cuts within the 3'-overhang, to generate a 33-nucleotide DNA that CC is probably incorporated into the CRISPR leader by a cut-and-paste CC mechanism (PubMed:26478180). This subunit's probable nuclease CC activity is not required for spacer acquisition (PubMed:24793649). CC {ECO:0000269|PubMed:21255106, ECO:0000269|PubMed:24793649, CC ECO:0000269|PubMed:24920831, 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 CC dimers sandwiching a Cas2 dimer (PubMed:24793649). The DNA lies CC across a flat surface extending from 1 Cas1 dimer, across the Cas2 CC dimer and contacting the other Cas1 dimer. Only 1 Cas1 protein CC from each dimer is catalytic, the other interacts with the Cas2 CC dimer and possibly target DNA (PubMed:26478180, PubMed:26503043). CC {ECO:0000269|PubMed:24793649, ECO:0000269|PubMed:24920831, CC ECO:0000269|PubMed:25707795, ECO:0000269|PubMed:26478180, CC ECO:0000269|PubMed:26503043, ECO:0000305|Ref.10, CC ECO:0000305|Ref.12}. 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 CC the 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 CC that generate a surface for DNA-binding across the Cas2 dimer and CC formation 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 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 ProteinModelPortal; P45956; -. DR SMR; P45956; 1-94. DR BioGrid; 4259585; 137. DR DIP; DIP-12109N; -. DR IntAct; P45956; 2. DR STRING; 511145.b2754; -. DR PaxDb; P45956; -. DR EnsemblBacteria; AAC75796; AAC75796; b2754. DR EnsemblBacteria; BAE76831; BAE76831; BAE76831. DR GeneID; 947213; -. DR KEGG; ecj:JW5438; -. DR KEGG; eco:b2754; -. DR PATRIC; 32120918; VBIEscCol129921_2851. DR EchoBASE; EB2694; -. DR EcoGene; EG12845; ygbF. DR eggNOG; ENOG4105PWE; Bacteria. DR eggNOG; ENOG4111VHR; LUCA. DR HOGENOM; HOG000015873; -. DR InParanoid; P45956; -. DR KO; K09951; -. DR OMA; GENRRMP; -. DR BioCyc; EcoCyc:EG12845-MONOMER; -. DR BioCyc; ECOL316407:JW5438-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; Complete proteome; DNA-binding; KW Endonuclease; Hydrolase; Nuclease; Reference proteome. FT CHAIN 1 94 CRISPR-associated endoribonuclease Cas2. FT /FTId=PRO_0000169312. FT MUTAGEN 9 9 E->A,R: No effect on spacer acquisition, FT Cas1-Cas2 complex formation or CRISPR FT DNA-binding by complex. FT {ECO:0000269|PubMed:24793649, FT ECO:0000269|PubMed:25707795}. FT MUTAGEN 10 10 N->A: No effect on spacer acquisition. FT {ECO:0000269|PubMed:24793649}. FT MUTAGEN 14 16 RLR->ALA: No in vivspacer acquisition, FT significantly decreased protospacer FT binding. {ECO:0000269|PubMed:26478180}. FT MUTAGEN 14 14 R->A: Slight decrease in spacer FT acquisition. FT {ECO:0000269|PubMed:24793649}. FT MUTAGEN 16 16 R->A: Slight decrease in spacer FT acquisition. FT {ECO:0000269|PubMed:24793649}. FT MUTAGEN 16 16 R->E: Dramatically decreased spacer FT acquisition in vivo. FT {ECO:0000269|PubMed:26503043}. FT MUTAGEN 18 18 R->A: Very little spacer acquisition. FT {ECO:0000269|PubMed:24793649}. FT MUTAGEN 27 27 R->A: Slight decrease in spacer FT acquisition. FT {ECO:0000269|PubMed:24793649}. FT MUTAGEN 38 40 KIR->AIA: Very little in vivo spacer FT acquisition. FT {ECO:0000269|PubMed:26478180}. FT MUTAGEN 65 65 E->A: No effect on spacer acquisition. FT {ECO:0000269|PubMed:24793649}. FT MUTAGEN 65 65 E->R: Slight decrease in spacer FT acquisition, Cas1-Cas2 complex formation FT or CRISPR DNA-binding by complex. Loss of FT spacer acquisition; when associated with FT R-84. {ECO:0000269|PubMed:24793649}. FT MUTAGEN 77 78 RR->AA: No spacer acquisition, FT significantly decreased protospacer FT binding. {ECO:0000269|PubMed:26478180}. FT MUTAGEN 77 77 R->E: No change in spacer acquisition in FT vivo. {ECO:0000269|PubMed:26503043}. FT MUTAGEN 78 78 R->E: Dramatically decreased spacer FT acquisition in vivo. FT {ECO:0000269|PubMed:26503043}. FT MUTAGEN 79 94 Missing: Loss of spacer acquisition, no FT Cas1-Cas2 complex formation, loss of FT CRISPR DNA-binding by complex (beta6- FT beta7 deletion). FT {ECO:0000269|PubMed:24793649, FT ECO:0000269|PubMed:25707795}. FT MUTAGEN 84 84 D->A: No effect on spacer acquisition. FT {ECO:0000269|PubMed:24793649}. FT MUTAGEN 84 84 D->R: Slight decrease in spacer FT acquisition. Loss of spacer acquisition; FT when associated with R-65. FT {ECO:0000269|PubMed:24793649}. FT STRAND 3 10 {ECO:0000244|PDB:4MAK}. FT HELIX 13 22 {ECO:0000244|PDB:4MAK}. FT STRAND 23 27 {ECO:0000244|PDB:4MAK}. FT STRAND 30 34 {ECO:0000244|PDB:4MAK}. FT HELIX 37 50 {ECO:0000244|PDB:4MAK}. FT STRAND 55 61 {ECO:0000244|PDB:4MAK}. FT STRAND 63 66 {ECO:0000244|PDB:4MAK}. FT STRAND 68 73 {ECO:0000244|PDB:4MAK}. FT STRAND 79 83 {ECO:0000244|PDB:4P6I}. FT STRAND 86 91 {ECO:0000244|PDB:4P6I}. SQ SEQUENCE 94 AA; 10518 MW; D1C159D924B477B4 CRC64; MSMLVVVTEN VPPRLRGRLA IWLLEVRAGV YVGDVSAKIR EMIWEQIAGL AEEGNVVMAW ATNTETGFEF QTFGLNRRTP VDLDGLRLVS FLPV //