Conserved residues in the delta subunit help the E. coli clamp loader, gamma complex, target primer-template DNA for clamp assembly.

Nucleic Acids Research
Siying ChenManju M Hingorani

Abstract

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 revea...Continue Reading

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Citations

Nov 20, 2012·The Journal of Biological Chemistry·Jaclyn N Hayner, Linda B Bloom
Mar 21, 2009·Annual Review of Biochemistry·Samir M Hamdan, Charles C Richardson
Jul 7, 2009·Biochimica Et Biophysica Acta·Zhihao Zhuang, Yongxing Ai
Mar 17, 2009·Journal of Molecular Biology·Siying ChenManju M Hingorani
Dec 27, 2011·Journal of Molecular Biology·Miho SakatoManju M Hingorani

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Methods Mentioned

BETA
column chromatography
gel-filtration
Fluorescence

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