The clamp loader assembles the beta clamp onto either a 3' or 5' primer terminus: the underlying basis favoring 3' loading.

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 gamma complex clamp loader can load the beta 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 beta 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 beta clamp facilitates its own loading specificity for a 3' primed site. Implications to eukaryotic clamp loader complexes are proposed.