This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Clamp loader proteins catalyze assembly of circular sliding clamps on DNA to enable processive DNA replication. During the reaction, the clamp loader binds primer-template DNA and positions it in the center of a clamp to form a topological link between the two. Clamp loaders are multiprotein complexes, such as the five protein Escherichia coli, Saccharomyces cerevisiae, and human clamp loaders, and the two protein Pyrococcus furiosus and Methanobacterium thermoautotrophicum clamp loaders, and thus far the site(s) responsible for binding and selecting primer-template DNA as the target for clamp assembly remain unknown. To address this issue, we analyzed the interaction between the E. coli g complex clamp loader and DNA using UV-induced protein?DNA cross-linking and mass spectrometry. The results show that the d subunit in the g complex makes close contact with the primer-template junction. Tryptophan 279 in the d Cterminal domain lies near the 30-OH primer end and may play a key role in primer-template recognition. Previous studies have shown that d also binds and opens the b clamp (hydrophobic residues in the N-terminal domain of d contact b. The clamp-binding and DNA-binding sites on d appear positioned for facile entry of primer-template into the center of the clamp and exit of the template strand from the complex. A similar analysis of the S. cerevisiae RFC complex suggests that the dual functionality observed for d in the g complex may be true also for clamp loaders from other organisms.
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