Essential amino acids of Escherichia coli DnaC protein in an N-terminal domain interact with DnaB helicase.
Escherichia coli DnaC protein bound to ATP forms a complex with DnaB protein. To identify the domain of DnaC that interacts with DnaB, a genetic selection was used based on the lethal effect of induced dnaC expression and a model that inviability arises by the binding of DnaC to DnaB to inhibit replication fork movement. The analysis of dnaC alleles that preserved viability under elevated expression revealed an N-terminal domain of DnaC involved in binding to DnaB. Mutant proteins bearing single amino acid substitutions (R10P, L11Q, L29Q, S41P, W32G, and L44P) that reside in regions of predicted secondary structure were inert in DNA replication activity because of their inability to bind to DnaB, but they retained ATP binding activity, as indicated by UV cross-linking to [alpha-(32)P]ATP. These alleles also failed to complement a dnaC28 mutant. Other selected mutations that map to regions carrying Walker A and B boxes are expected to be defective in ATP binding, a required step in DnaB-DnaC complex formation. Lastly, we found that the sixth codon from the N terminus encodes aspartate, resolving a reported discrepancy between the predicted amino acid sequence based on DNA sequencing data and the results from N-terminal amino acid sequencing (Nakayama, N., Bond, M. W., Miyajima, A., Kobori, J., and Arai, K. (1987) J. Biol. Chem. 262, 10475-10480).
Ludlam
A V
AV
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319, USA.
McNatt
M W
MW
Carr
K M
KM
Kaguni
J M
JM
D000483:Alleles; D000595:Amino Acid Sequence; D000601:Amino Acids, Essential / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004265:DNA Helicases / Q000378:metabolism*; D053488:DnaB Helicases; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005804:Genes, Lethal; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017386:Sequence Homology, Amino Acid
D000601:Amino Acids, Essential; D001426:Bacterial Proteins; C071702:DnaC protein, E coli; D029968:Escherichia coli Proteins; D004265:DNA Helicases; D053488:DnaB Helicases