Journal of Biological Chemistry
Volume 284, Issue 45, 6 November 2009, Pages 31473-31483
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DNA: Replication, Repair, Recombination, and Chromosome Dynamics
The Clamp Loader Assembles the β Clamp onto Either a 3′ or 5′ Primer Terminus: THE UNDERLYING BASIS FAVORING 3′ LOADING*

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Clamp loaders assemble sliding clamps onto 3′ primed sites for DNA polymerases. Clamp loaders are thought to be specific for a 3′ primed site, and unable to bind a 5′ site. We demonstrate here that the Escherichia coli γ complex clamp loader can load the β clamp onto a 5′ primed site, although with at least 20-fold reduced efficiency relative to loading at a 3′ primed site. Preferential clamp loading at a 3′ site does not appear to be due to DNA binding, as the clamp loader forms an avid complex with β at a 5′ site. Preferential loading at a 3′ versus a 5′ site occurs at the ATP hydrolysis step, needed to close the ring around DNA. We also address DNA structural features that are recognized for preferential loading at a 3′ site. Although the single-stranded template strand extends in opposite directions from 3′ and 5′ primed sites, thus making it a favorite candidate for distinguishing between 3′ and 5′ sites, the single-strand polarity at a primed template junction does not determine 3′ site selection for clamp loading. Instead, we find that clamp loader recognition of a 3′ site lies in the duplex portion of the primed site, not the single-strand portion. We present evidence that the β clamp facilitates its own loading specificity for a 3′ primed site. Implications to eukaryotic clamp loader complexes are proposed.

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*

This work was supported, in whole or in part, by National Institutes of Health Grant GM3883.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Figs. S1–S8.