Delineation of structural domains and identification of functionally important residues in DNA repair enzyme exonuclease VII.
Exonuclease VII (ExoVII) is a bacterial nuclease involved in DNA repair and recombination that hydrolyses single-stranded DNA. ExoVII is composed of two subunits: large XseA and small XseB. Thus far, little was known about the molecular structure of ExoVII, the interactions between XseA and XseB, the architecture of the nuclease active site or its mechanism of action. We used bioinformatics methods to predict the structure of XseA, which revealed four domains: an N-terminal OB-fold domain, a middle putatively catalytic domain, a coiled-coil domain and a short C-terminal segment. By series of deletion and site-directed mutagenesis experiments on XseA from Escherichia coli, we determined that the OB-fold domain is responsible for DNA binding, the coiled-coil domain is involved in binding multiple copies of the XseB subunit and residues D155, R205, H238 and D241 of the middle domain are important for the catalytic activity but not for DNA binding. Altogether, we propose a model of sequence-structure-function relationships in ExoVII.
Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology, ks. Trojdena 4, PL-02-109 Warsaw, Poland.
Katarzyna
K
Poleszak
Katarzyna H
KH
Kaminska
Stanislaw
S
Dunin-Horkawicz
Andrei
A
Lupas
Krzysztof J
KJ
Skowronek
Janusz M
JM
Bujnicki
D000595:Amino Acid Sequence; D001483:Base Sequence; D045643:DNA Repair Enzymes / Q000737:chemistry* / Q000145:classification / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000145:classification / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000737:chemistry* / Q000145:classification / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D029968:Escherichia coli Proteins; D021122:Protein Subunits; D005090:Exodeoxyribonucleases; C576363:XseA protein, E coli; C026870:exodeoxyribonuclease VII; D045643:DNA Repair Enzymes