CAS1_ECOLI
ID CAS1_ECOLI Reviewed; 305 AA.
AC Q46896; Q2MA74;
DT 01-JUN-2001, integrated into UniProtKB/Swiss-Prot.
DT 01-NOV-1996, sequence version 1.
DT 03-AUG-2022, entry version 137.
DE RecName: Full=CRISPR-associated endonuclease Cas1;
DE EC=3.1.-.-;
GN Name=ygbT; Synonyms=cas1; OrderedLocusNames=b2755, JW2725;
OS Escherichia coli (strain K12).
OC Bacteria; Proteobacteria; Gammaproteobacteria; Enterobacterales;
OC Enterobacteriaceae; Escherichia.
OX NCBI_TaxID=83333;
RN [1]
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 [2]
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 [3]
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 [4]
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 [5]
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 [6]
RP FUNCTION AS A SPACER INTEGRASE, SUBUNIT, AND MUTAGENESIS OF ASP-221.
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 [7]
RP FUNCTION AS A SPACER INTEGRASE, PROBABLE REACTION MECHANISM, COFACTOR,
RP SUBUNIT, AND MUTAGENESIS OF TYR-149; TYR-165; TYR-188; HIS-208; TYR-217;
RP ASP-221 AND LYS-224.
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 [8]
RP FUNCTION, AND COFACTOR.
RC STRAIN=K12 / MG1655 / ATCC 47076;
RX PubMed=26284603; DOI=10.7554/elife.08716;
RA Rollie C., Schneider S., Brinkmann A.S., Bolt E.L., White M.F.;
RT "Intrinsic sequence specificity of the Cas1 integrase directs new spacer
RT acquisition.";
RL Elife 4:0-0(2015).
RN [9]
RP X-RAY CRYSTALLOGRAPHY (1.95 ANGSTROMS), X-RAY CRYSTALLOGRAPHY (1.4
RP ANGSTROMS) OF 92-291, FUNCTION AS AN ENDONUCLEASE, COFACTOR, ACTIVITY
RP REGULATION, SUBUNIT, INTERACTION WITH RECB; RECC; RUVB; CASC AND CASE,
RP SUBCELLULAR LOCATION, DISRUPTION PHENOTYPE, AND MUTAGENESIS OF GLU-141;
RP THR-184; TYR-188; HIS-208; LYS-211; ASP-218; ASP-221 AND LYS-224.
RC STRAIN=K12;
RX PubMed=21219465; DOI=10.1111/j.1365-2958.2010.07465.x;
RA Babu M., Beloglazova N., Flick R., Graham C., Skarina T., Nocek B.,
RA Gagarinova A., Pogoutse O., Brown G., Binkowski A., Phanse S.,
RA Joachimiak A., Koonin E.V., Savchenko A., Emili A., Greenblatt J.,
RA Edwards A.M., Yakunin A.F.;
RT "A dual function of the CRISPR-Cas system in bacterial antivirus immunity
RT and DNA repair.";
RL Mol. Microbiol. 79:484-502(2011).
RN [10]
RP X-RAY CRYSTALLOGRAPHY (2.3 ANGSTROMS) IN COMPLEX WITH CAS2, FUNCTION IN
RP SPACER ACQUISITION, INTERACTION WITH CAS2 (YGBF), SUBUNIT, DNA-BINDING, AND
RP MUTAGENESIS OF GLU-141; HIS-208; ASP-218; ASP-221; LYS-224; ARG-245;
RP ARG-252; ARG-256; 282-PRO--SER-305 AND ILE-291.
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 [11]
RP X-RAY CRYSTALLOGRAPHY (2.70 ANGSTROMS) IN COMPLEX WITH CAS2, 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 [12]
RP X-RAY CRYSTALLOGRAPHY (2.60 ANGSTROMS) OF 2-281 IN COMPLEX WITH CAS2 AND
RP DNA, FUNCTION, SUBUNIT, DOMAIN, DNA-BINDING, AND MUTAGENESIS OF TYR-22;
RP ARG-59; ARG-84; TYR-165; TRP-170; TYR-188; HIS-208; TYR-217;
RP 245-ARG--ARG-248 AND 256-ARG--LYS-259.
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 [13] {ECO:0007744|PDB:5DS4, ECO:0007744|PDB:5DS5, ECO:0007744|PDB:5DS6}
RP X-RAY CRYSTALLOGRAPHY (2.95 ANGSTROMS) IN COMPLEX WITH MAGNESIUM; CAS2 AND
RP DNA, FUNCTION, SUBUNIT, DNA-BINDING, AND MUTAGENESIS OF TYR-22; ARG-41;
RP ARG-59; ARG-66; ARG-84; ARG-245 AND ARG-248.
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). Non-specifically binds DNA; the Cas1-Cas2 complex
CC preferentially binds CRISPR-locus DNA (PubMed:24793649). Highest
CC binding is seen to a dual forked DNA complex with 3'-overhangs and a
CC 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). Cas1 alone
CC endonucleolytically cleaves linear ssRNA, ssDNA and short (34 base)
CC dsDNA as well as branched DNA substrates such as Holliday junctions,
CC replication forks and 5'-flap DNA substrates (PubMed:21219465). In
CC vitro catalyzes a concerted transesterification reaction on branched
CC DNA, as would be expected during integration of protospacers into the
CC CRISPR leader sequence; Cas2 is not required in vitro. This reaction
CC requires a 3'-OH group at the branch point (PubMed:26284603). Genetic
CC interactions suggest Cas1 interacts with components of the RecBC and
CC RuvB DNA repair systems (PubMed:21219465).
CC {ECO:0000269|PubMed:21219465, ECO:0000269|PubMed:21255106,
CC ECO:0000269|PubMed:24793649, ECO:0000269|PubMed:24920831,
CC ECO:0000269|PubMed:25707795, ECO:0000269|PubMed:26284603,
CC ECO:0000269|PubMed:26478180}.
CC -!- COFACTOR:
CC Name=Mg(2+); Xref=ChEBI:CHEBI:18420;
CC Evidence={ECO:0000269|PubMed:21219465, ECO:0000269|PubMed:25707795,
CC ECO:0000269|PubMed:26284603};
CC Note=Protospacer integration in vitro also occurs with Mn(2+) and also
CC requires low concentrations of KCl (PubMed:25707795). The
CC transesterification function works equally well with Mg(2+), Mn(2+) or
CC Co(2+) in vitro (PubMed:26284603). {ECO:0000269|PubMed:25707795,
CC ECO:0000269|PubMed:26284603};
CC -!- ACTIVITY REGULATION: Nuclease activity partially inhibited by CasE
CC (PubMed:21219465). {ECO:0000269|PubMed:21219465}.
CC -!- SUBUNIT: Homodimer (PubMed:21219465). Part of the Cas1-Cas2 complex
CC (PubMed:24920831, PubMed:24793649, PubMed:25707795, Ref.11,
CC PubMed:26478180, PubMed:26503043). Interacts with RecB, RecC, RuvB,
CC CasC and CasE (PubMed:21219465). Forms a hexamer with 2 Cas1 dimers
CC sandwiching a Cas2 dimer (PubMed:24793649, PubMed:26478180). The DNA
CC lies across a flat surface extending from 1 Cas1 dimer, across the Cas2
CC dimer and contacting the other Cas1 dimer. Only 1 Cas1 protein from
CC each dimer is catalytic, the other interacts with the Cas2 dimer and
CC possibly target DNA (PubMed:26478180, PubMed:26503043).
CC {ECO:0000269|PubMed:21219465, 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.11}.
CC -!- INTERACTION:
CC Q46896; P76339: hprS; NbExp=3; IntAct=EBI-1130209, EBI-554869;
CC Q46896; P45956: ygbF; NbExp=8; IntAct=EBI-1130209, EBI-9150552;
CC Q46896; Q46896: ygbT; NbExp=4; IntAct=EBI-1130209, EBI-1130209;
CC -!- SUBCELLULAR LOCATION: Cytoplasm {ECO:0000269|PubMed:21219465}. Note=In
CC 15% of cell localizes to discrete nucleoid foci (probable DNA damage
CC sites) upon treatment with mitomycin C (MMC) for 2 hours
CC (PubMed:21219465). {ECO:0000269|PubMed:21219465}.
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: Not essential. Increased sensitivity to MMC and
CC UV light; double ygbT-ruvA, ruvB or ruvC disruptions have no further
CC phenotype suggesting Cas1 functions in the same DNA repair pathway
CC (PubMed:21219465). Function in DNA repair also seems to require CRISPRs
CC (PubMed:21219465). Cells elongate after 2 hours growth in MMC; they are
CC even longer in double ygbT-ruvA, ruvB or ruvC disruptions, suggesting
CC Cas1 may also function in chromosome segregation (PubMed:21219465).
CC Loss of plasmid silencing (PubMed:21255106).
CC {ECO:0000269|PubMed:21219465, ECO:0000269|PubMed:21255106}.
CC -!- SIMILARITY: Belongs to the CRISPR-associated endonuclease Cas1 family.
CC {ECO:0000305}.
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DR EMBL; U29579; AAA69265.1; -; Genomic_DNA.
DR EMBL; U00096; AAC75797.1; -; Genomic_DNA.
DR EMBL; AP009048; BAE76832.1; -; Genomic_DNA.
DR PIR; G65056; G65056.
DR RefSeq; NP_417235.1; NC_000913.3.
DR RefSeq; WP_000220066.1; NZ_LN832404.1.
DR PDB; 3NKD; X-ray; 1.95 A; A/B=1-305.
DR PDB; 3NKE; X-ray; 1.40 A; A/B/C=92-291.
DR PDB; 4P6I; X-ray; 2.30 A; C/D/E/F=1-305.
DR PDB; 4QDL; X-ray; 2.70 A; A/B/C/D=1-305.
DR PDB; 5DLJ; X-ray; 2.60 A; A/B/C/D=2-281.
DR PDB; 5DQT; X-ray; 3.10 A; A/B/C/D/I/J/K/L=1-305.
DR PDB; 5DQU; X-ray; 4.50 A; A/B/C/D=1-305.
DR PDB; 5DQZ; X-ray; 2.70 A; A/B/C/D=1-305.
DR PDB; 5DS4; X-ray; 3.20 A; A/B/C/D=1-305.
DR PDB; 5DS5; X-ray; 2.95 A; A/B/C/D=1-305.
DR PDB; 5DS6; X-ray; 3.35 A; A/B/C/D=1-305.
DR PDB; 5VVJ; X-ray; 3.89 A; A/B/C/D=1-305.
DR PDB; 5VVK; X-ray; 2.90 A; A/B/C/D=1-305.
DR PDB; 5VVL; X-ray; 3.31 A; A/B/C/D=1-305.
DR PDB; 5WFE; EM; 3.64 A; A/B/C/D=1-305.
DR PDBsum; 3NKD; -.
DR PDBsum; 3NKE; -.
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; Q46896; -.
DR SMR; Q46896; -.
DR BioGRID; 4261582; 279.
DR BioGRID; 851560; 13.
DR ComplexPortal; CPX-996; Cas1-Cas2 complex.
DR DIP; DIP-12118N; -.
DR IntAct; Q46896; 16.
DR STRING; 511145.b2755; -.
DR PaxDb; Q46896; -.
DR PRIDE; Q46896; -.
DR EnsemblBacteria; AAC75797; AAC75797; b2755.
DR EnsemblBacteria; BAE76832; BAE76832; BAE76832.
DR GeneID; 947228; -.
DR KEGG; ecj:JW2725; -.
DR KEGG; eco:b2755; -.
DR PATRIC; fig|1411691.4.peg.3983; -.
DR EchoBASE; EB2915; -.
DR eggNOG; COG1518; Bacteria.
DR HOGENOM; CLU_077904_1_0_6; -.
DR InParanoid; Q46896; -.
DR OMA; IWVGEAG; -.
DR PhylomeDB; Q46896; -.
DR BioCyc; EcoCyc:G7425-MON; -.
DR PRO; PR:Q46896; -.
DR Proteomes; UP000000318; Chromosome.
DR Proteomes; UP000000625; Chromosome.
DR GO; GO:0005737; C:cytoplasm; IEA:UniProtKB-SubCell.
DR GO; GO:0017108; F:5'-flap endonuclease activity; IDA:EcoCyc.
DR GO; GO:0008821; F:crossover junction endodeoxyribonuclease activity; IDA:EcoCyc.
DR GO; GO:0003677; F:DNA binding; IEA:UniProtKB-KW.
DR GO; GO:0042802; F:identical protein binding; IPI:IntAct.
DR GO; GO:0046872; F:metal ion binding; IEA:UniProtKB-UniRule.
DR GO; GO:0006974; P:cellular response to DNA damage stimulus; IMP:EcoCyc.
DR GO; GO:0099048; P:CRISPR-cas system; IDA:ComplexPortal.
DR GO; GO:0051607; P:defense response to virus; IEA:UniProtKB-UniRule.
DR GO; GO:0006281; P:DNA repair; IEA:UniProtKB-KW.
DR GO; GO:0043571; P:maintenance of CRISPR repeat elements; IDA:ComplexPortal.
DR CDD; cd09719; Cas1_I-E; 1.
DR Gene3D; 1.20.120.920; -; 1.
DR Gene3D; 3.100.10.20; -; 1.
DR HAMAP; MF_01470; Cas1; 1.
DR InterPro; IPR033641; Cas1_I-E.
DR InterPro; IPR002729; CRISPR-assoc_Cas1.
DR InterPro; IPR042206; CRISPR-assoc_Cas1_C.
DR InterPro; IPR019851; CRISPR-assoc_Cas1_ECOLI.
DR InterPro; IPR042211; CRISPR-assoc_Cas1_N.
DR Pfam; PF01867; Cas_Cas1; 1.
DR TIGRFAMs; TIGR00287; cas1; 2.
DR TIGRFAMs; TIGR03638; cas1_ECOLI; 1.
PE 1: Evidence at protein level;
KW 3D-structure; Antiviral defense; Cytoplasm; DNA damage; DNA repair;
KW DNA-binding; Endonuclease; Hydrolase; Magnesium; Metal-binding; Nuclease;
KW Reference proteome.
FT CHAIN 1..305
FT /note="CRISPR-associated endonuclease Cas1"
FT /id="PRO_0000169315"
FT REGION 96..278
FT /note="Sufficient for cleavage of ssRNA, ssDNA and Holliday
FT junction DNA"
FT REGION 278..305
FT /note="Disordered"
FT /evidence="ECO:0000256|SAM:MobiDB-lite"
FT BINDING 141
FT /ligand="Mg(2+)"
FT /ligand_id="ChEBI:CHEBI:18420"
FT /evidence="ECO:0000269|PubMed:26503043,
FT ECO:0007744|PDB:5DS5"
FT BINDING 208
FT /ligand="Mg(2+)"
FT /ligand_id="ChEBI:CHEBI:18420"
FT /evidence="ECO:0000305|PubMed:26503043"
FT BINDING 221
FT /ligand="Mg(2+)"
FT /ligand_id="ChEBI:CHEBI:18420"
FT /evidence="ECO:0000269|PubMed:26503043,
FT ECO:0007744|PDB:5DS5"
FT MUTAGEN 22
FT /note="Y->A: Slightly decreased spacer acquisition in
FT vivo."
FT /evidence="ECO:0000269|PubMed:26478180,
FT ECO:0000269|PubMed:26503043"
FT MUTAGEN 22
FT /note="Y->F: Nearly wild-type spacer acquisition in vivo."
FT /evidence="ECO:0000269|PubMed:26503043"
FT MUTAGEN 41
FT /note="R->E: Dramatically decreased spacer acquisition in
FT vivo."
FT /evidence="ECO:0000269|PubMed:26503043"
FT MUTAGEN 59
FT /note="R->A: Loss of spacer acquisition in vivo, decreased
FT protospacer binding."
FT /evidence="ECO:0000269|PubMed:26478180"
FT MUTAGEN 59
FT /note="R->D: Dramatically decreased spacer acquisition in
FT vitro, 250-fold decreased affinity for protospacer DNA."
FT /evidence="ECO:0000269|PubMed:26503043"
FT MUTAGEN 66
FT /note="R->D: Dramatically decreased spacer acquisition in
FT vitro, 250-fold decreased affinity for protospacer DNA."
FT /evidence="ECO:0000269|PubMed:26503043"
FT MUTAGEN 66
FT /note="R->E: Dramatically decreased spacer acquisition in
FT vivo."
FT /evidence="ECO:0000269|PubMed:26503043"
FT MUTAGEN 84
FT /note="R->A: Decreased spacer acquisition in vivo."
FT /evidence="ECO:0000269|PubMed:26478180"
FT MUTAGEN 84
FT /note="R->E: Dramatically decreased spacer acquisition in
FT vivo."
FT /evidence="ECO:0000269|PubMed:26503043"
FT MUTAGEN 141
FT /note="E->A: No cleavage of any substrates, no restoration
FT of UV or mitomycin C (MMC) resistance (PubMed:21219465).
FT Loss of spacer acquisition in vivo (PubMed:24793649)."
FT /evidence="ECO:0000269|PubMed:21219465,
FT ECO:0000269|PubMed:24793649"
FT MUTAGEN 149
FT /note="Y->A: No effect on in vitro protospacer
FT integration."
FT /evidence="ECO:0000269|PubMed:25707795"
FT MUTAGEN 165
FT /note="Y->A: No effect on in vitro protospacer integration
FT (PubMed:25707795). Alone significantly decreased
FT protospacer acquisition in vivo (PubMed:26478180). Loss of
FT protospacer acquisition, decreased protospacer binding; in
FT association with A-170, significantly decreased protospacer
FT binding; in association with A-217 (PubMed:26478180)."
FT /evidence="ECO:0000269|PubMed:25707795,
FT ECO:0000269|PubMed:26478180"
FT MUTAGEN 170
FT /note="W->A: Alone significantly decreased protospacer
FT acquisition in vivo (PubMed:26478180). Decreased
FT protospacer binding; in association with A-170
FT (PubMed:26478180)."
FT /evidence="ECO:0000269|PubMed:26478180"
FT MUTAGEN 184
FT /note="T->A: No cleavage of any substrates."
FT /evidence="ECO:0000269|PubMed:21219465"
FT MUTAGEN 188
FT /note="Y->A: Partial inhibition of cleavage
FT (PubMed:21219465). No effect on in vitro protospacer
FT integration (PubMed:25707795). Significantly decreased
FT protospacer acquisition in vivo (PubMed:26478180)."
FT /evidence="ECO:0000269|PubMed:21219465,
FT ECO:0000269|PubMed:25707795, ECO:0000269|PubMed:26478180"
FT MUTAGEN 208
FT /note="H->A: No cleavage of any substrates, no restoration
FT of UV or MMC resistance (PubMed:21219465). Loss of spacer
FT acquisition in vivo (PubMed:24793649, PubMed:25707795,
FT PubMed:26478180)."
FT /evidence="ECO:0000269|PubMed:21219465,
FT ECO:0000269|PubMed:24793649, ECO:0000269|PubMed:25707795,
FT ECO:0000269|PubMed:26478180"
FT MUTAGEN 211
FT /note="K->A: No cleavage of any substrates."
FT /evidence="ECO:0000269|PubMed:21219465"
FT MUTAGEN 217
FT /note="Y->A: No effect on in vitro protospacer integration
FT (PubMed:25707795). Alone significantly decreased
FT protospacer acquisition in vivo (PubMed:26478180).
FT Significantly decreased protospacer binding; in association
FT with A-165 (PubMed:26478180)."
FT /evidence="ECO:0000269|PubMed:25707795,
FT ECO:0000269|PubMed:26478180"
FT MUTAGEN 218
FT /note="D->A: No cleavage of any substrates, no restoration
FT of UV or MMC resistance (PubMed:21219465). Loss of spacer
FT acquisition in vivo (PubMed:24793649)."
FT /evidence="ECO:0000269|PubMed:21219465,
FT ECO:0000269|PubMed:24793649"
FT MUTAGEN 221
FT /note="D->A: No cleavage of any substrates
FT (PubMed:21219465). Loss of spacer acquisition in vivo
FT (PubMed:24793649, PubMed:24920831, PubMed:25707795). No
FT cleavage of CRISPR leader in preparation for spacer
FT integration (PubMed:24920831)."
FT /evidence="ECO:0000269|PubMed:21219465,
FT ECO:0000269|PubMed:24793649, ECO:0000269|PubMed:24920831,
FT ECO:0000269|PubMed:25707795"
FT MUTAGEN 224
FT /note="K->A: No cleavage of any substrates
FT (PubMed:21219465). Loss of spacer acquisition in vivo
FT (PubMed:24793649, PubMed:25707795)."
FT /evidence="ECO:0000269|PubMed:21219465,
FT ECO:0000269|PubMed:24793649, ECO:0000269|PubMed:25707795"
FT MUTAGEN 245..248
FT /note="REVR->AEVA: Loss of spacer acquisition in vivo."
FT /evidence="ECO:0000269|PubMed:26478180"
FT MUTAGEN 245
FT /note="R->A: No effect on spacer acquisition."
FT /evidence="ECO:0000269|PubMed:24793649"
FT MUTAGEN 245
FT /note="R->D: Decreased spacer acquisition."
FT /evidence="ECO:0000269|PubMed:24793649"
FT MUTAGEN 245
FT /note="R->E: Dramatically decreased spacer acquisition in
FT vivo."
FT /evidence="ECO:0000269|PubMed:26503043"
FT MUTAGEN 248
FT /note="R->E: Dramatically decreased spacer acquisition in
FT vivo."
FT /evidence="ECO:0000269|PubMed:26503043"
FT MUTAGEN 252
FT /note="R->A: No effect on spacer acquisition."
FT /evidence="ECO:0000269|PubMed:24793649"
FT MUTAGEN 252
FT /note="R->E: Loss of spacer acquisition, no Cas1-Cas2
FT complex formation, loss of CRISPR DNA-binding by complex.
FT Protein is stable and dimerizes."
FT /evidence="ECO:0000269|PubMed:24793649"
FT MUTAGEN 256..259
FT /note="RSSK->ASSA: Loss of spacer acquisition in vivo."
FT /evidence="ECO:0000269|PubMed:26478180"
FT MUTAGEN 256
FT /note="R->A: No effect on spacer acquisition."
FT /evidence="ECO:0000269|PubMed:24793649"
FT MUTAGEN 256
FT /note="R->E: Loss of spacer acquisition."
FT /evidence="ECO:0000269|PubMed:24793649"
FT MUTAGEN 282..305
FT /note="Missing: No effect on spacer acquisition, Cas1-Cas2
FT complex formation or CRISPR DNA-binding by complex."
FT /evidence="ECO:0000269|PubMed:24793649"
FT MUTAGEN 291
FT /note="I->G,R: No effect on spacer acquisition."
FT /evidence="ECO:0000269|PubMed:24793649"
FT HELIX 11..13
FT /evidence="ECO:0007829|PDB:5DLJ"
FT STRAND 17..20
FT /evidence="ECO:0007829|PDB:3NKD"
FT STRAND 22..28
FT /evidence="ECO:0007829|PDB:3NKD"
FT STRAND 31..36
FT /evidence="ECO:0007829|PDB:3NKD"
FT STRAND 37..39
FT /evidence="ECO:0007829|PDB:4P6I"
FT STRAND 41..43
FT /evidence="ECO:0007829|PDB:3NKD"
FT HELIX 46..48
FT /evidence="ECO:0007829|PDB:3NKD"
FT STRAND 50..54
FT /evidence="ECO:0007829|PDB:3NKD"
FT STRAND 58..60
FT /evidence="ECO:0007829|PDB:3NKD"
FT HELIX 62..70
FT /evidence="ECO:0007829|PDB:3NKD"
FT STRAND 74..79
FT /evidence="ECO:0007829|PDB:3NKD"
FT HELIX 80..82
FT /evidence="ECO:0007829|PDB:3NKD"
FT STRAND 85..90
FT /evidence="ECO:0007829|PDB:3NKD"
FT HELIX 95..107
FT /evidence="ECO:0007829|PDB:3NKE"
FT HELIX 109..124
FT /evidence="ECO:0007829|PDB:3NKE"
FT STRAND 130..132
FT /evidence="ECO:0007829|PDB:4QDL"
FT HELIX 134..156
FT /evidence="ECO:0007829|PDB:3NKE"
FT HELIX 167..169
FT /evidence="ECO:0007829|PDB:5DS5"
FT HELIX 170..172
FT /evidence="ECO:0007829|PDB:4P6I"
FT HELIX 175..197
FT /evidence="ECO:0007829|PDB:3NKE"
FT STRAND 206..208
FT /evidence="ECO:0007829|PDB:3NKE"
FT HELIX 214..223
FT /evidence="ECO:0007829|PDB:3NKE"
FT TURN 224..227
FT /evidence="ECO:0007829|PDB:3NKE"
FT HELIX 228..238
FT /evidence="ECO:0007829|PDB:3NKE"
FT HELIX 243..258
FT /evidence="ECO:0007829|PDB:3NKE"
FT HELIX 260..273
FT /evidence="ECO:0007829|PDB:3NKE"
FT STRAND 275..277
FT /evidence="ECO:0007829|PDB:5DQT"
SQ SEQUENCE 305 AA; 33194 MW; 01A31BA98453D8A5 CRC64;
MTWLPLNPIP LKDRVSMIFL QYGQIDVIDG AFVLIDKTGI RTHIPVGSVA CIMLEPGTRV
SHAAVRLAAQ VGTLLVWVGE AGVRVYASGQ PGGARSDKLL YQAKLALDED LRLKVVRKMF
ELRFGEPAPA RRSVEQLRGI EGSRVRATYA LLAKQYGVTW NGRRYDPKDW EKGDTINQCI
SAATSCLYGV TEAAILAAGY APAIGFVHTG KPLSFVYDIA DIIKFDTVVP KAFEIARRNP
GEPDREVRLA CRDIFRSSKT LAKLIPLIED VLAAGEIQPP APPEDAQPVA IPLPVSLGDA
GHRSS
