Nucleic Acids Research Advance Access published online on April 19, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn157
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Nucleic acid Enzymes |
Conserved residues in the 
subunit help the E. coli clamp loader, 
complex, target primer-template DNA for clamp assembly
1Molecular Biology and Biochemistry Department, Wesleyan University, Middletown, CT 06459 and 2The Rockefeller University, New York, NY 10021, USA
*To whom correspondence should be addressed. Tel: +01 860 685 2284; Fax: +01 860 685 2141; Email: mhingorani{at}wesleyan.edu
Received December 4, 2007. Revised March 6, 2008. Accepted March 19, 2008.
The Escherichia coli clamp loader, 
complex (3
'
), catalyzes ATP-driven assembly of β clamps onto primer-template DNA (p/tDNA), enabling processive replication. The mechanism by which 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 complex and clamp assembly, but only at far higher concentrations than p/tDNA. Thus, contact between 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—HRVW279QNRR—in subunit contributes to such interactions, since Tryptophan-279 specifically cross-links to the primer-template junction. Mutation of -W279 weakens complex binding to p/tDNA, hampering its ability to load clamps and promote proccessive DNA replication, and additional mutations in the sequence (-R277, -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.