REP75_PYRAB
ID REP75_PYRAB Reviewed; 654 AA.
AC O54003;
DT 28-NOV-2012, integrated into UniProtKB/Swiss-Prot.
DT 01-JUN-1998, sequence version 1.
DT 03-AUG-2022, entry version 50.
DE RecName: Full=Replication initiator protein;
DE Short=Rep75 protein;
DE EC=2.7.7.31;
DE EC=3.1.21.-;
DE EC=6.5.1.1;
DE AltName: Full=ORF1;
GN Name=rep75; Synonyms=orf1;
OS Pyrococcus abyssi (strain GE5 / Orsay).
OG Plasmid pGT5.
OC Archaea; Euryarchaeota; Thermococci; Thermococcales; Thermococcaceae;
OC Pyrococcus.
OX NCBI_TaxID=272844;
RN [1]
RP NUCLEOTIDE SEQUENCE [GENOMIC DNA], FUNCTION, COFACTOR, BIOPHYSICOCHEMICAL
RP PROPERTIES, AND DNA-BINDING.
RC STRAIN=GE5 / Orsay;
RX PubMed=9570403; DOI=10.1046/j.1365-2958.1998.00759.x;
RA Marsin S., Forterre P.;
RT "A rolling circle replication initiator protein with a nucleotidyl-
RT transferase activity encoded by the plasmid pGT5 from the hyperthermophilic
RT archaeon Pyrococcus abyssi.";
RL Mol. Microbiol. 27:1183-1192(1998).
RN [2]
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=GE5 / Orsay;
RX PubMed=8655503; DOI=10.1128/jb.178.11.3232-3237.1996;
RA Erauso G., Marsin S., Benbouzid-Rollet N., Baucher M.F., Barbeyron T.,
RA Zivanovic Y., Prieur D., Forterre P.;
RT "Sequence of plasmid pGT5 from the archaeon Pyrococcus abyssi: evidence for
RT rolling-circle replication in a hyperthermophile.";
RL J. Bacteriol. 178:3232-3237(1996).
RN [3]
RP FUNCTION, BIOPHYSICOCHEMICAL PROPERTIES, ACTIVITY REGULATION, DNA
RP SEQUENCE-SPECIFICITY, AND MUTAGENESIS OF TYR-448 AND ARG-451.
RC STRAIN=GE5 / Orsay;
RX PubMed=10417644; DOI=10.1046/j.1365-2958.1999.01498.x;
RA Marsin S., Forterre P.;
RT "The active site of the rolling circle replication protein Rep75 is
RT involved in site-specific nuclease, ligase and nucleotidyl transferase
RT activities.";
RL Mol. Microbiol. 33:537-545(1999).
RN [4]
RP FUNCTION AS A TOPOISOMERASE, AND MUTAGENESIS OF TYR-448 AND ARG-451.
RC STRAIN=GE5 / Orsay;
RX PubMed=10871346; DOI=10.1093/nar/28.11.2251;
RA Marsin S., Marguet E., Forterre P.;
RT "Topoisomerase activity of the hyperthermophilic replication initiator
RT protein Rep75.";
RL Nucleic Acids Res. 28:2251-2255(2000).
CC -!- FUNCTION: Required for rolling circle plasmid replication, has a site-
CC specific endonuclease/ligase activity (nicking and closing). In vitro
CC (no in vivo system yet exists) cleaves the double-stranded origin of
CC replication (dso) site, on an ssDNA template, remaining covalently
CC linked to the 5' end. Religates the appropriate substrates. Has
CC nucleotidyltransferase activity, adding ATP or dATP to the 3' end of
CC the nicked ssDNA site. Both activities require a G nucleotide at the 3'
CC end of the nicking site. Also has topoisomerase activity, nicking and
CC relaxing negatively supercoiled plasmids in a narrow concentration
CC range (25-50 molar ratio of protein:DNA). Topoisomerase is not
CC dependent on a dso sequence. Has no topoisomerase activity on slightly
CC positively supercoiled plasmid. {ECO:0000269|PubMed:10417644,
CC ECO:0000269|PubMed:10871346, ECO:0000269|PubMed:9570403}.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=a 2'-deoxyribonucleoside 5'-triphosphate + DNA(n) =
CC diphosphate + DNA(n+1); Xref=Rhea:RHEA:22508, Rhea:RHEA-COMP:17339,
CC Rhea:RHEA-COMP:17340, ChEBI:CHEBI:33019, ChEBI:CHEBI:61560,
CC ChEBI:CHEBI:173112; EC=2.7.7.31;
CC -!- CATALYTIC ACTIVITY:
CC Reaction=ATP + (deoxyribonucleotide)n-3'-hydroxyl + 5'-phospho-
CC (deoxyribonucleotide)m = (deoxyribonucleotide)n+m + AMP +
CC diphosphate.; EC=6.5.1.1;
CC -!- COFACTOR:
CC Name=Mn(2+); Xref=ChEBI:CHEBI:29035;
CC Evidence={ECO:0000269|PubMed:9570403};
CC Note=DNA nicking and nucleotidyltransferase are strictly dependent on
CC Mn(2+); Mg(2+) is able to substitute in DNA nicking 5X less
CC efficiently. {ECO:0000269|PubMed:9570403};
CC -!- ACTIVITY REGULATION: ATP and dATP inhibit nicking and closing while
CC stimulating nucleotidyltransferase. {ECO:0000269|PubMed:10417644}.
CC -!- BIOPHYSICOCHEMICAL PROPERTIES:
CC Temperature dependence:
CC Optimum temperature is 105 degrees Celsius for ssDNA nicking, 75
CC degrees Celsius for nucleotidyltransferase activity on a ssDNA
CC substrate. Optimum temperature for ssDNA closing is substrate
CC dependent, being 55 and 75 degrees Celsius for the 2 substrates
CC tested. Topoisomerase activity is only seen between 55 and 75 degrees
CC Celsius. {ECO:0000269|PubMed:10417644, ECO:0000269|PubMed:9570403};
CC -!- PTM: The N-terminus is blocked when overexpressed in E.coli.
CC -!- MISCELLANEOUS: Plasmids from hyperthermophilic archaea are relaxed to
CC positively supercoiled at physiological temperatures.
CC -!- SIMILARITY: Belongs to the Gram-positive plasmids replication protein
CC type 1 family. {ECO:0000305}.
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DR EMBL; AJ002587; CAA05626.1; -; Genomic_DNA.
DR EMBL; U49503; -; NOT_ANNOTATED_CDS; Genomic_DNA.
DR AlphaFoldDB; O54003; -.
DR PRIDE; O54003; -.
DR eggNOG; ENOG502N5AJ; Archaea.
DR BRENDA; 2.7.7.31; 5242.
DR Proteomes; UP000000810; Plasmid pGT5.
DR GO; GO:0005524; F:ATP binding; IEA:UniProtKB-KW.
DR GO; GO:0003677; F:DNA binding; IEA:UniProtKB-KW.
DR GO; GO:0003910; F:DNA ligase (ATP) activity; IEA:UniProtKB-EC.
DR GO; GO:0003912; F:DNA nucleotidylexotransferase activity; IEA:UniProtKB-EC.
DR GO; GO:0003916; F:DNA topoisomerase activity; IEA:UniProtKB-KW.
DR GO; GO:0004519; F:endonuclease activity; IEA:UniProtKB-KW.
DR GO; GO:0006260; P:DNA replication; IEA:UniProtKB-KW.
DR GO; GO:0006276; P:plasmid maintenance; IEA:UniProtKB-KW.
PE 1: Evidence at protein level;
KW ATP-binding; Coiled coil; DNA replication; DNA-binding; Endonuclease;
KW Hydrolase; Isomerase; Ligase; Nuclease; Nucleotide-binding; Plasmid;
KW Plasmid copy control; Topoisomerase; Transferase.
FT CHAIN 1..654
FT /note="Replication initiator protein"
FT /id="PRO_0000420230"
FT COILED 535..585
FT /evidence="ECO:0000255"
FT ACT_SITE 448
FT /note="O-(5'-phospho-DNA)-tyrosine intermediate"
FT /evidence="ECO:0000305"
FT MUTAGEN 448
FT /note="Y->F: Loss of nicking and closing activities, binds,
FT not covalently, to ssDNA. Still has nucleotidyltransferase
FT activity. Loss of topoisomerase activity. Loss of all
FT activities; when associated with L-451."
FT /evidence="ECO:0000269|PubMed:10417644,
FT ECO:0000269|PubMed:10871346"
FT MUTAGEN 451
FT /note="R->L: Loss of nucleotidyltransferase activity,
FT reduced DNA closing but no loss of ssDNA nicking. Decreased
FT plasmid nicking, no closing activity on supercoiled
FT plasmids. Loss of all activities; when associated with F-
FT 448."
FT /evidence="ECO:0000269|PubMed:10417644,
FT ECO:0000269|PubMed:10871346"
SQ SEQUENCE 654 AA; 75224 MW; 16635E4BBDA50BB4 CRC64;
MVIYTSKFKN SLLDGLGVGH LSYDDQPILC NEVHPTLTLD TFISGGSSGS RPRPRWVYLD
ISTTNEGISE EFSSNTESKS PLLKVCGVSS KSSEGDGSSF IWFDKYRSVI SRLEHSGFVE
VERTVLKLRK ISKELRSINK QLSRLFLDDR ERAKLLSRKR KYLDFARALI GSISKSLTLY
ADRFFVEVPQ DYAKLIQKLG FSSDTLLLHL FVNSGVLEVF LDDNSSHKFR IAYISKVHAG
KYHPVKGISK GSQEAKRVLR DLLVLSELLE GSLVSYRSGG VETIHHLIPV RHFVLTAPKE
LSFSIWASLK KGDSSLFRAF KDAGAKAIKE FLSYLASKEH ISGNLLFGFT INVHVTGDKN
PFEPHFHIDA IVTFICYDKS STKWFRLNPL LSESDLKKLR DIWKNVLLSY FGELLSEDTK
SKDFDVWAGD NYYSLPLDVP QVFFELKYAS RKLFVNFVNY FEQSNFDESS VSDWDFVRFV
FEYSNRTERY GFLTNIKRYL SMSCSHLVEK RVQELEEFIS RIEFDLSVNG NKMSDSLKRA
LLERLEYLKD ELSELKERGF EYLFERALEK AEELLSNDNL TLERVIHILE TLFTALGKSI
VNYNFYVELE DVSFREFVDY LYDNHLSDVL VFSDRHRSIT IIRLIPPPDG GVPV
