Characterizing the conformational dynamics for DNA loading of the Escherichia coli DNA polymerase III subunit beta clamp
Authors: Liriano Melissa Lizette , Koleva Bilyana , Beuning Penny J 
Published: June 2021

In Escherichia coli, the replicative polymerase, DNA polymerase III (Pol III), is made up of ten proteins, making up three subassemblies: polymerase III core, β sliding clamp, and the loader complex. In the presence of the β sliding clamp, replication progresses at a rate of >50 kb per association event, relative to 10 nucleotides per event in its absence. The β sliding clamp is, therefore, required to ensure that replication is efficient and processive. The solved crystal structure of β shows that it is composed of two crescent‐shaped protomers, and each unit consists of three domains. The two protomers combine in a head‐to‐tail orientation to form a homodimer ring structure. For replication to commence, the β sliding clamp must be loaded onto double‐stranded DNA by the multiprotein subunit, the loader complex (δγ3δ′). The loading of β requires at least a two‐step mechanism, which includes a binding event between β and the loader complex and opening of the β ring at the dimer interface. However, the exact mode of ring opening is not clearly understood. One mechanistic model proposes that ATP binding at two γ domain interfaces induces a conformational change in the clamp loading complex facilitating the N‐terminal end of the δ subunit to bind to the C‐terminal face of the β sliding clamp. In a wrench‐like action, the δ subunit of the loader complex opens the β clamp by dissociating Domain I from Domain III and then β is loaded onto the primed DNA duplex. With the β sliding clamp loaded, ATP hydrolysis is stimulated, causing the clamp to close, restoring the dimeric clamp ring structure and ejecting the loader complex. However, our previous work showed that the β sliding clamp is not a rigid, closed structure in solution but rather that it is dynamic and likely able to open spontaneously. To further investigate the dynamics of the β clamp interacting with the loader complex, we made mutations in the highly dynamic Domain I and at the dimer interface of the β clamp. These mutations have either stabilized or destabilized clamp loading onto DNA. We have also designed, expressed, and purified a β clamp made up of two protomers with the same amino acid sequence but different DNA codons. This has allowed us to selectively mutate residues in exactly one protomer. In this variant, one dimer interface is fused with an 18‐amino acid‐long linker that restricts interactions with the one free interface without hindering the clamp's biochemical or cellular activities. Loading assays have been performed to assess the efficiency of β loading, by the loader complex, onto a primer:template DNA for each of these variants. Reaction products were detected and quantified with SDS‐PAGE and Western blots, respectively, revealing differences in clamp loading efficiency for stabilized and destabilized clamp variants.Support or Funding InformationNIH R01GM123239This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.