OLD_THESS
ID OLD_THESS Reviewed; 528 AA.
AC E8PLM2;
DT 03-AUG-2022, integrated into UniProtKB/Swiss-Prot.
DT 05-APR-2011, sequence version 1.
DT 03-AUG-2022, entry version 49.
DE RecName: Full=OLD nuclease {ECO:0000303|PubMed:32009148};
DE EC=3.1.11.3 {ECO:0000269|PubMed:32009148};
DE AltName: Full=Exodeoxyribonuclease OLD {ECO:0000305};
DE AltName: Full=Overcoming lysogenization defect {ECO:0000303|PubMed:32009148};
DE Short=Old {ECO:0000303|PubMed:32009148};
GN Name=old {ECO:0000303|PubMed:32009148}; OrderedLocusNames=TSC_c04750;
OS Thermus scotoductus (strain ATCC 700910 / SA-01).
OC Bacteria; Deinococcus-Thermus; Deinococci; Thermales; Thermaceae; Thermus.
OX NCBI_TaxID=743525;
RN [1] {ECO:0000312|EMBL:ADW21107.1}
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=ATCC 700910 / SA-01;
RX PubMed=22115438; DOI=10.1186/1471-2164-12-577;
RA Gounder K., Brzuszkiewicz E., Liesegang H., Wollherr A., Daniel R.,
RA Gottschalk G., Reva O., Kumwenda B., Srivastava M., Bricio C.,
RA Berenguer J., van Heerden E., Litthauer D.;
RT "Sequence of the hyperplastic genome of the naturally competent Thermus
RT scotoductus SA-01.";
RL BMC Genomics 12:577-577(2011).
RN [2] {ECO:0007744|PDB:6P74}
RP X-RAY CRYSTALLOGRAPHY (2.20 ANGSTROMS) IN COMPLEX WITH METAL AND ATP
RP ANALOG, FUNCTION, CATALYTIC ACTIVITY, PROBABLE ACTIVE SITE, COFACTOR,
RP BIOPHYSICOCHEMICAL PROPERTIES, SUBUNIT, DOMAIN, ATP-BINDING, AND
RP MUTAGENESIS OF LYS-34; HIS-140; GLU-276; HIS-283; HIS-310; GLU-377;
RP ASP-381; ASP-431; ASP-433; SER-478; GLU-480 AND ARG-487.
RC STRAIN=ATCC 700910 / SA-01;
RX PubMed=32009148; DOI=10.1093/nar/gkaa059;
RA Schiltz C.J., Adams M.C., Chappie J.S.;
RT "The full-length structure of Thermus scotoductus OLD defines the ATP
RT hydrolysis properties and catalytic mechanism of Class 1 OLD family
RT nucleases.";
RL Nucleic Acids Res. 48:2762-2776(2020).
CC -!- FUNCTION: An exodeoxyribonuclease that degrades linear or supercoiled
CC dsDNA from 5'-3'. Nicks and linearizes circular DNA. Activity is not
CC stimulated by ATP or AMP-PNP, although it has DNA-stimulated ATPase
CC activity. {ECO:0000269|PubMed:32009148}.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=Exonucleolytic cleavage in the 5'- to 3'-direction to yield
CC nucleoside 5'-phosphates.; EC=3.1.11.3;
CC Evidence={ECO:0000269|PubMed:32009148};
CC -!- COFACTOR:
CC Name=Mg(2+); Xref=ChEBI:CHEBI:18420;
CC Evidence={ECO:0000269|PubMed:32009148};
CC Name=Mn(2+); Xref=ChEBI:CHEBI:29035;
CC Evidence={ECO:0000269|PubMed:32009148};
CC Name=Ca(2+); Xref=ChEBI:CHEBI:29108;
CC Evidence={ECO:0000269|PubMed:32009148};
CC Note=Probably binds 2 metal cations. In vitro during a short
CC incubation, Mn(2+) is most efficient on linear or supercoiled dsDNA,
CC nicks but only poorly digests dsDNA with Co(2+), Ni(2+) or Zn(2+). When
CC purified from E.coli Ca(2+) and Mg(2+) are the most abundant metals.
CC {ECO:0000269|PubMed:32009148};
CC -!- BIOPHYSICOCHEMICAL PROPERTIES:
CC Kinetic parameters:
CC KM=180 uM for ATP {ECO:0000269|PubMed:32009148};
CC Note=kcat is 0.44 min(-1) for ATPase at 65 degrees Celsius.
CC {ECO:0000269|PubMed:32009148};
CC Temperature dependence:
CC Optimum temperature is 65 degrees Celsius for the ATPase activity.
CC {ECO:0000269|PubMed:32009148};
CC -!- SUBUNIT: Homodimer. {ECO:0000269|PubMed:32009148}.
CC -!- DOMAIN: The ATPase domain dimerizes on its own via its dimerization
CC region, the Toprim (topoisomerase/primase) domain does not. The Toprim
CC domain has Mn(2+)-dependent nuclease activity on linear and supercoiled
CC dsDNA. It faces away from the dimerization domain.
CC {ECO:0000269|PubMed:32009148}.
CC -!- SIMILARITY: Belongs to the class 1 OLD nuclease family.
CC {ECO:0000305|PubMed:32009148}.
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DR EMBL; CP001962; ADW21107.1; -; Genomic_DNA.
DR PDB; 6P74; X-ray; 2.20 A; A=1-528.
DR PDBsum; 6P74; -.
DR SMR; E8PLM2; -.
DR STRING; 743525.TSC_c04750; -.
DR EnsemblBacteria; ADW21107; ADW21107; TSC_c04750.
DR KEGG; tsc:TSC_c04750; -.
DR eggNOG; COG1196; Bacteria.
DR eggNOG; COG3593; Bacteria.
DR HOGENOM; CLU_017618_1_0_0; -.
DR Proteomes; UP000008087; Chromosome.
DR GO; GO:0004519; F:endonuclease activity; IEA:UniProtKB-KW.
DR CDD; cd01026; TOPRIM_OLD; 1.
DR Gene3D; 3.40.50.300; -; 1.
DR InterPro; IPR041685; AAA_15.
DR InterPro; IPR027417; P-loop_NTPase.
DR InterPro; IPR034139; TOPRIM_OLD.
DR Pfam; PF13175; AAA_15; 2.
DR SUPFAM; SSF52540; SSF52540; 1.
PE 1: Evidence at protein level;
KW 3D-structure; ATP-binding; Exonuclease; Hydrolase; Metal-binding; Nuclease;
KW Nucleotide-binding.
FT CHAIN 1..528
FT /note="OLD nuclease"
FT /id="PRO_0000456031"
FT REGION 1..153
FT /note="ATPase domain N-terminus"
FT /evidence="ECO:0000305|PubMed:32009148"
FT REGION 154..245
FT /note="Dimerization domain"
FT /evidence="ECO:0000305|PubMed:32009148"
FT REGION 246..369
FT /note="ATPase domain C-terminus"
FT /evidence="ECO:0000305|PubMed:32009148"
FT REGION 370..528
FT /note="Toprim domain"
FT /evidence="ECO:0000305|PubMed:32009148"
FT ACT_SITE 487
FT /note="Stabilizes transition state or protonates leaving
FT group"
FT /evidence="ECO:0000305|PubMed:32009148"
FT BINDING 31..35
FT /ligand="ATP"
FT /ligand_id="ChEBI:CHEBI:30616"
FT /evidence="ECO:0000305|PubMed:32009148"
FT BINDING 377
FT /ligand="a divalent metal cation"
FT /ligand_id="ChEBI:CHEBI:60240"
FT /ligand_label="1"
FT /evidence="ECO:0000305|PubMed:32009148"
FT BINDING 381
FT /ligand="a divalent metal cation"
FT /ligand_id="ChEBI:CHEBI:60240"
FT /ligand_label="2"
FT /evidence="ECO:0000305|PubMed:32009148"
FT BINDING 431
FT /ligand="a divalent metal cation"
FT /ligand_id="ChEBI:CHEBI:60240"
FT /ligand_label="1"
FT /evidence="ECO:0000305|PubMed:32009148"
FT BINDING 433
FT /ligand="a divalent metal cation"
FT /ligand_id="ChEBI:CHEBI:60240"
FT /ligand_label="1"
FT /evidence="ECO:0000305|PubMed:32009148"
FT BINDING 478
FT /ligand="a divalent metal cation"
FT /ligand_id="ChEBI:CHEBI:60240"
FT /ligand_label="2"
FT /evidence="ECO:0000305|PubMed:32009148"
FT BINDING 480
FT /ligand="a divalent metal cation"
FT /ligand_id="ChEBI:CHEBI:60240"
FT /ligand_label="2"
FT /evidence="ECO:0000305|PubMed:32009148"
FT MUTAGEN 34
FT /note="K->A: Loss of ATPase, no change in exonuclease
FT activity."
FT /evidence="ECO:0000269|PubMed:32009148"
FT MUTAGEN 140
FT /note="H->A: Loss of ATPase."
FT /evidence="ECO:0000269|PubMed:32009148"
FT MUTAGEN 140
FT /note="H->Q: 2.4-fold decrease in ATPase."
FT /evidence="ECO:0000269|PubMed:32009148"
FT MUTAGEN 276
FT /note="E->A: Loss of ATPase, no change in exonuclease
FT activity."
FT /evidence="ECO:0000269|PubMed:32009148"
FT MUTAGEN 283
FT /note="H->A: 4-fold decrease in ATPase, no change in
FT exonuclease activity."
FT /evidence="ECO:0000269|PubMed:32009148"
FT MUTAGEN 283
FT /note="H->D: Nearly complete loss of ATPase."
FT /evidence="ECO:0000269|PubMed:32009148"
FT MUTAGEN 310
FT /note="H->A: Loss of ATPase."
FT /evidence="ECO:0000269|PubMed:32009148"
FT MUTAGEN 377
FT /note="E->A: No change in exonuclease or nicking activity.
FT Loss of exonuclease but not nicking; when associated with
FT A-431 and A-433."
FT /evidence="ECO:0000269|PubMed:32009148"
FT MUTAGEN 381
FT /note="D->A: No change in exonuclease or nicking activity.
FT Loss of exonuclease but not nicking; when associated with
FT A-478 and A-480."
FT /evidence="ECO:0000269|PubMed:32009148"
FT MUTAGEN 431
FT /note="D->A: No change in exonuclease or nicking activity.
FT Loss of exonuclease but not nicking; when associated with
FT A-377 and A-433. Loss of exonuclease and nicking; when
FT associated with A-433; A-478 and A-480."
FT /evidence="ECO:0000269|PubMed:32009148"
FT MUTAGEN 433
FT /note="D->A: No change in exonuclease or nicking activity.
FT Loss of exonuclease but not nicking; when associated with
FT A-377 and A-431. Loss of exonuclease and nicking; when
FT associated with A-431; A-478 and A-480."
FT /evidence="ECO:0000269|PubMed:32009148"
FT MUTAGEN 478
FT /note="S->A: No change in exonuclease or nicking activity.
FT Loss of exonuclease but not nicking; when associated with
FT A-381 and A-480. Loss of exonuclease and nicking; when
FT associated with A-431; A-433 and A-480."
FT /evidence="ECO:0000269|PubMed:32009148"
FT MUTAGEN 480
FT /note="E->A: No change in exonuclease or nicking activity.
FT Loss of exonuclease but not nicking; when associated with
FT A-381 and A-478. Loss of exonuclease and nicking; when
FT associated with A-431; A-433 and A-478."
FT /evidence="ECO:0000269|PubMed:32009148"
FT MUTAGEN 487
FT /note="R->A: Reduced exonuclease, reduced nicking
FT activity."
FT /evidence="ECO:0000269|PubMed:32009148"
FT MUTAGEN 487
FT /note="R->E: Loss of exonuclease, reduced nicking
FT activity."
FT /evidence="ECO:0000269|PubMed:32009148"
SQ SEQUENCE 528 AA; 59748 MW; BE11A4B6ACDB0647 CRC64;
MLKRLQVKNF RCLEDIDLPL GPLTAIVGPN GAGKTTILRA IDLVLGDVWP SLRSFRIPQD
FINFDTTRAI EITVHFDPPY TQGSFNITAF RLTCKGEDAD FHVDLEPLDE GGNVPRYPSG
NPLRVGTDMR NHARVLFLDH RRNLAQHLPS IRGSILGRLL QPVRREFKLQ DNFKQVYEQA
MDLLRTEQVK QIEKTIAETA KQMLGFLGKD AMKSMEIGFG FADPANPFNS LRLQYRESDL
TLPGDELGLG IQSAIVVGIF EAFRQLGEKI GTVIIEEPEM YLHPQAQRYF YRLLCEMADK
DQCQIIYSTH SPIFADVNRF EALRLVRKDR DDRVVVSYVR EEDKSALDNV RNRFKLGGRF
DTARNEVLFA KRALLVEGYG DRVAALQLFN QLEVDPDAEC IAVVDCGGKA GIELIVGVCK
ALDIPFVVVH DEDVWPIDER ADEETRRKQE QENKAEQEKN QRIQACAGAE RVFVVQPSLE
AALGIGRNAS DKPYRIAEIL KTVDVGQPPD ALRPFVEAIR QVTRPMEE
