Conserved residues in the delta subunit help the E. coli clamp loader, gamma complex, target primer-template DNA for clamp assembly.
The Escherichia coli clamp loader, gamma complex (gamma(3)deltadelta'lambdapsi), catalyzes ATP-driven assembly of beta clamps onto primer-template DNA (p/tDNA), enabling processive replication. The mechanism by which gamma complex targets p/tDNA for clamp assembly is not resolved. According to previous studies, charged/polar amino acids inside the clamp loader chamber interact with the double-stranded (ds) portion of p/tDNA. We find that dsDNA, not ssDNA, can trigger a burst of ATP hydrolysis by gamma complex and clamp assembly, but only at far higher concentrations than p/tDNA. Thus, contact between gamma complex and dsDNA is necessary and sufficient, but not optimal, for the reaction, and additional contacts with p/tDNA likely facilitate its selection as the optimal substrate for clamp assembly. We investigated whether a conserved sequence-HRVW(279)QNRR--in delta subunit contributes to such interactions, since Tryptophan-279 specifically cross-links to the primer-template junction. Mutation of delta-W279 weakens gamma complex binding to p/tDNA, hampering its ability to load clamps and promote proccessive DNA replication, and additional mutations in the sequence (delta-R277, delta-R283) worsen the interaction. These data reveal a novel location in the C-terminal domain of the E. coli clamp loader that contributes to DNA binding and helps define p/tDNA as the preferred substrate for the reaction.
Chen
Siying
S
Molecular Biology and Biochemistry Department, Wesleyan University, Middletown, CT 06459 and The Rockefeller University, New York, NY 10021, USA.
Coman
Maria Magdalena
MM
Sakato
Miho
M
O'Donnell
Michael
M
Hingorani
Manju M
MM
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism*; D004258:DNA Polymerase III / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000378:metabolism; D004261:DNA Replication; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D013698:Templates, Genetic; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics
D017931:DNA Primers; D029968:Escherichia coli Proteins; D014364:Tryptophan; D000255:Adenosine Triphosphate; D004247:DNA; C101139:beta subunit, DNA polymerase III; D004258:DNA Polymerase III; D000251:Adenosine Triphosphatases