Hexameric RSF1010 helicase RepA: the structural and functional importance of single amino acid residues.
In the known monoclinic crystals the 3-dimensional structure of the hexameric, replicative helicase RepA encoded by plasmid RSF1010 shows 6-fold rotational symmetry. In contrast, in the cubic crystal form at 2.55 A resolution described here RepA has 3-fold symmetry and consists of a trimer of dimers. To study structure-function relationships, a series of repA deletion mutants and mutations yielding single amino acid exchanges were constructed and the respective gene products were analyzed in vivo and in vitro. Hexamerization of RepA occurs via the N-terminus and is required for NTP hydrolysis. The C-terminus is essential both for the interaction with the replication machinery and for the helicase activity. Functional analyses of RepA variants with single amino acid exchanges confirmed most of the predictions that were based on the published 3-dimensional structure. Of the five motifs conserved in family 4 helicases, all residues conserved in RepA and T7 gp4 helicases participate in DNA unwinding. Residues K42, E76, D77, D139 and H178, proposed to play key roles in catalyzing the hydrolysis of NTPs, are essential for RepA activity. Residue H178 of motif H3 couples nucleotide consumption to DNA strand separation.
Ziegelin
Günter
G
Max-Planck-Institut für Molekulare Genetik, Ihnestrasse 73, Dahlem, D-14195 Berlin, Germany.
Niedenzu
Timo
T
Lurz
Rudi
R
Saenger
Wolfram
W
Lanka
Erich
E
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000409:Alanine / Q000235:genetics; D000483:Alleles; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication / Q000235:genetics; D004268:DNA-Binding Proteins; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005816:Genetic Complementation Test; D005973:Glutamine / Q000235:genetics; D008969:Molecular Sequence Data; D010641:Phenotype; D010957:Plasmids / Q000235:genetics*; D017354:Point Mutation; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators
D004268:DNA-Binding Proteins; D011506:Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D005973:Glutamine; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; D000409:Alanine