In <i>Escherichia coli</i>, the beta sliding clamp is an essential subunit of the replicative polymerase DNA polymerase III (Pol III). In the presence of the beta sliding clamp, the efficiency and processivity of DNA replication is significantly enhanced, with a rate of &gt;50 kb added versus 10 nucleotides in the absence of the clamp. The beta sliding clamp is a homodimeric ring structure, with monomers arranged in a head-to-tail orientation yielding two identical interfaces. Loading of the beta sliding clamp onto DNA requires opening of the clamp ring, which is facilitated by a second multiprotein subunit of the replisome called the clamp loader, or the gamma complex. The gamma clamp loader complex is composed of three proteins: delta, delta prime, and gamma. While a partially opened beta is presumably required for beta loading onto DNA by the gamma complex, the exact clamp opening mechanism remains unknown. One model proposes that the delta subunit opens the beta clamp, in a wrench-like action, dissociating Domain I in one monomer from Domain III of the other monomer. We previously showed that the beta sliding clamp is highly dynamic in Domain I, transiently sampling conformational states in solution. We have made mutations at the dimer interface and have probed the stability of each variant and assessed their DNA loading efficiencies. We have also designed and purified a beta clamp variant that has one interface fused with an 18-amino acid long linker. This linked beta variant is homodimeric but each monomer has a distinct DNA sequence, allowing us to make mutations in only one monomer. The linked beta variant supports many beta functions; thus, we can gain more insight into how restricting interactions to one interface of the beta clamp affects its thermostability, interactions with the gamma complex, and DNA loading efficiency.

Probing the thermostability and DNA loading efficiency of beta sliding clamp variants with perturbed dimer interfaces

2019 Fall National Meeting

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