DART_THEA5
ID DART_THEA5 Reviewed; 222 AA.
AC P0DV56;
DT 03-AUG-2022, integrated into UniProtKB/Swiss-Prot.
DT 03-AUG-2022, sequence version 1.
DT 03-AUG-2022, entry version 1.
DE RecName: Full=DNA ADP-ribosyl transferase {ECO:0000303|PubMed:27939941};
DE Short=DarT {ECO:0000303|PubMed:27939941};
DE EC=2.4.2.- {ECO:0000269|PubMed:27939941, ECO:0000269|PubMed:34408320};
DE AltName: Full=Toxin DarT {ECO:0000303|PubMed:27939941};
GN Name=darT {ECO:0000305}; ORFNames=TO73_1568 {ECO:0000312|EMBL:ALJ91409.1};
OS Thermus aquaticus (strain ATCC BAA-2747 / Y51MC23).
OC Bacteria; Deinococcus-Thermus; Deinococci; Thermales; Thermaceae; Thermus.
OX NCBI_TaxID=498848;
RN [1]
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=BAA-2747 / Y51MC23;
RX PubMed=26465632; DOI=10.1371/journal.pone.0138674;
RA Brumm P.J., Monsma S., Keough B., Jasinovica S., Ferguson E.,
RA Schoenfeld T., Lodes M., Mead D.A.;
RT "Complete Genome Sequence of Thermus aquaticus Y51MC23.";
RL PLoS ONE 10:e0138674-e0138674(2015).
RN [2]
RP FUNCTION AS A TOXIN, FUNCTION AS AN ADP-RIBOSYL TRANSFERASE, SUBSTRATE
RP SPECIFICITY, CATALYTIC ACTIVITY, EXPRESSION IN E.COLI, SUBUNIT, PROBABLE
RP ACTIVE SITE, AND MUTAGENESIS OF GLU-160.
RX PubMed=27939941; DOI=10.1016/j.molcel.2016.11.014;
RA Jankevicius G., Ariza A., Ahel M., Ahel I.;
RT "The Toxin-Antitoxin System DarTG Catalyzes Reversible ADP-Ribosylation of
RT DNA.";
RL Mol. Cell 64:1109-1116(2016).
RN [3]
RP FUNCTION AS A TOXIN, FUNCTION AS AN ADP-RIBOSYL TRANSFERASE, CATALYTIC
RP ACTIVITY, POSSIBLE REACTION MECHANISM, ACTIVE SITE, EXPRESSION IN E.COLI,
RP AND MUTAGENESIS OF TYR-47; HIS-53; ARG-54; HIS-68; TYR-74; ARG-78; MET-81;
RP TYR-83; HIS-122; ARG-154; GLN-158 AND GLU-160.
RX PubMed=34408320; DOI=10.1038/s41586-021-03825-4;
RA Schuller M., Butler R.E., Ariza A., Tromans-Coia C., Jankevicius G.,
RA Claridge T.D.W., Kendall S.L., Goh S., Stewart G.R., Ahel I.;
RT "Molecular basis for DarT ADP-ribosylation of a DNA base.";
RL Nature 596:597-602(2021).
CC -!- FUNCTION: Toxic component of a hybrid type II/IV toxin-antitoxin (TA)
CC system. Its toxic effect is neutralized by cognate antitoxin DarG. ADP-
CC ribosylates ssDNA on the second thymidine of the consensus sequence 5'-
CC TNTC-3'; the protein does not auto-modify. Has no activity on dsDNA in
CC vitro. This leads to a decrease in DNA replication. Upon expression in
CC E.coli inhibits cell growth, colony formation and induces the SOS
CC response. Expression leads to bacteriostasis; however if cells grow
CC over an hour in the presence of toxin, growth is no longer restored on
CC antitoxin-inducing plates (PubMed:27939941). In E.coli ADP-ribosylates
CC genomic DNA (gDNA), which induces RecA expression (a marker for DNA
CC damage) (PubMed:34408320). {ECO:0000269|PubMed:27939941,
CC ECO:0000269|PubMed:34408320}.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=a thymidine in DNA + NAD(+) = an N-(ADP-alpha-D-ribosyl)-
CC thymidine in DNA + H(+) + nicotinamide; Xref=Rhea:RHEA:71651,
CC Rhea:RHEA-COMP:13556, Rhea:RHEA-COMP:18051, ChEBI:CHEBI:15378,
CC ChEBI:CHEBI:17154, ChEBI:CHEBI:57540, ChEBI:CHEBI:137386,
CC ChEBI:CHEBI:191199;
CC PhysiologicalDirection=left-to-right; Xref=Rhea:RHEA:71652;
CC Evidence={ECO:0000269|PubMed:27939941, ECO:0000269|PubMed:34408320};
CC -!- SUBUNIT: Probably forms a complex with cognate antitoxin DarG; this
CC complex might decrease ADP-ribosylation activity of DarT.
CC {ECO:0000305|PubMed:27939941}.
CC -!- DOMAIN: The NAD(+)-binding element stabilizes the ADP-ribosylating
CC turn-turn (ARTT) loop which confers substrate specificity; both domains
CC contribute to ssDNA-binding. {ECO:0000250|UniProtKB:A0A0B0SG80}.
CC -!- SIMILARITY: Belongs to the DarT ADP-ribosyltransferase family.
CC {ECO:0000305}.
CC -!- SEQUENCE CAUTION:
CC Sequence=ALJ91409.1; Type=Erroneous initiation; Note=Truncated N-terminus.; Evidence={ECO:0000305};
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DR EMBL; CP010822; ALJ91409.1; ALT_INIT; Genomic_DNA.
PE 1: Evidence at protein level;
KW DNA-binding; Glycosyltransferase; Nucleotidyltransferase;
KW Toxin-antitoxin system; Transferase.
FT CHAIN 1..222
FT /note="DNA ADP-ribosyl transferase"
FT /id="PRO_0000456051"
FT DOMAIN 15..209
FT /note="DarT"
FT /evidence="ECO:0000255"
FT DNA_BIND 47..53
FT /evidence="ECO:0000250|UniProtKB:A0A0B0SG80"
FT DNA_BIND 78..83
FT /evidence="ECO:0000250|UniProtKB:A0A0B0SG80"
FT DNA_BIND 148..151
FT /evidence="ECO:0000250|UniProtKB:A0A0B0SG80"
FT DNA_BIND 154..158
FT /evidence="ECO:0000250|UniProtKB:A0A0B0SG80"
FT REGION 38..56
FT /note="NAD(+)-binding element"
FT /evidence="ECO:0000250|UniProtKB:A0A0B0SG80"
FT REGION 119..160
FT /note="ADP-ribosylating turn-turn loop"
FT /evidence="ECO:0000250|UniProtKB:A0A0B0SG80"
FT ACT_SITE 54
FT /note="Proton acceptor"
FT /evidence="ECO:0000305|PubMed:34408320"
FT ACT_SITE 160
FT /evidence="ECO:0000305|PubMed:27939941,
FT ECO:0000305|PubMed:34408320"
FT BINDING 16..18
FT /ligand="NAD(+)"
FT /ligand_id="ChEBI:CHEBI:57540"
FT /evidence="ECO:0000250|UniProtKB:A0A0B0SG80"
FT BINDING 54
FT /ligand="NAD(+)"
FT /ligand_id="ChEBI:CHEBI:57540"
FT /evidence="ECO:0000250|UniProtKB:A0A0B0SG80"
FT MUTAGEN 47
FT /note="Y->A: Still toxic in E.coli, decreased DNA ADP-
FT ribosylation activity."
FT /evidence="ECO:0000269|PubMed:34408320"
FT MUTAGEN 53
FT /note="H->A: Still toxic in E.coli, decreased DNA ADP-
FT ribosylation activity."
FT /evidence="ECO:0000269|PubMed:34408320"
FT MUTAGEN 54
FT /note="R->A,K: No longer toxic in E.coli, no longer ADP-
FT ribosylates ssDNA. No ADP-ribosylation of gDNA in vivo."
FT /evidence="ECO:0000269|PubMed:34408320"
FT MUTAGEN 68
FT /note="H->A: Still toxic in E.coli, decreased ADP-
FT ribosylation."
FT /evidence="ECO:0000269|PubMed:34408320"
FT MUTAGEN 74
FT /note="Y->A,S: Still toxic in E.coli, no longer ADP-
FT ribosylates ssDNA."
FT /evidence="ECO:0000269|PubMed:34408320"
FT MUTAGEN 74
FT /note="Y->F: Still toxic in E.coli, decreased ADP-
FT ribosylation."
FT /evidence="ECO:0000269|PubMed:34408320"
FT MUTAGEN 78
FT /note="R->A: Still toxic in E.coli, decreased DNA ADP-
FT ribosylation activity."
FT /evidence="ECO:0000269|PubMed:34408320"
FT MUTAGEN 81
FT /note="M->A: Still toxic in E.coli, no longer ADP-
FT ribosylates ssDNA."
FT /evidence="ECO:0000269|PubMed:34408320"
FT MUTAGEN 83
FT /note="Y->A: Still toxic in E.coli, decreased DNA ADP-
FT ribosylation activity."
FT /evidence="ECO:0000269|PubMed:34408320"
FT MUTAGEN 122
FT /note="H->A: No longer toxic in E.coli, no longer ADP-
FT ribosylates ssDNA. No ADP-ribosylation of gDNA in vivo."
FT /evidence="ECO:0000269|PubMed:34408320"
FT MUTAGEN 122
FT /note="H->N: Still toxic in E.coli, decreased ADP-
FT ribosylation."
FT /evidence="ECO:0000269|PubMed:34408320"
FT MUTAGEN 154
FT /note="R->A: Still toxic in E.coli, decreased DNA ADP-
FT ribosylation activity."
FT /evidence="ECO:0000269|PubMed:34408320"
FT MUTAGEN 154
FT /note="R->W: No longer toxic in E.coli, no longer ADP-
FT ribosylates ssDNA. No ADP-ribosylation of gDNA in vivo."
FT /evidence="ECO:0000269|PubMed:34408320"
FT MUTAGEN 158
FT /note="Q->A: Still toxic in vivo, decreased DNA ADP-
FT ribosylation activity."
FT /evidence="ECO:0000269|PubMed:34408320"
FT MUTAGEN 160
FT /note="E->A: No longer toxic when expressed in E.coli, no
FT longer ADP-ribosylates ssDNA, no effect on DNA replication.
FT No ADP-ribosylation of gDNA in vivo. Very low activity at
FT low DNA, high protein levels and after 24 hours."
FT /evidence="ECO:0000269|PubMed:27939941,
FT ECO:0000269|PubMed:34408320"
FT MUTAGEN 160
FT /note="E->D,Q: No longer toxic in E.coli, no longer ADP-
FT ribosylates ssDNA."
FT /evidence="ECO:0000269|PubMed:34408320"
SQ SEQUENCE 222 AA; 25702 MW; BC5ED363012570E6 CRC64;
MPQQGLAYPV PTLIYHITHL NNLQGILQRG GLLPYSQRPP TQQNVAYGHI QAHRAQVVVP
VGPRGKLHDY VPFYFCPRSP MLYAIHTQQT DYQGDQRPIL HLVSSAQKVA EARIPFVFTD
RHAAVQYVCF FHKLEHLKAL DWQAIQASYW ANVREKKQAE FLVKDFFPWE LVEEIGVIDK
TIQAQVESIL AQFPDLHHPP VRVRRSWYYK KRLCSASCEA TF
