Escherichia coli processivity clamp β from DNA polymerase III is dynamic in solution.


Escherichia coli DNA polymerase III is a highly processive replicase ...
Escherichia coli DNA polymerase III is a highly processive replicase because of the presence of the β clamp protein that tethers DNA polymerases to DNA. The β clamp is a head-to-tail ring-shaped homodimer, in which each protomer contains three structurally similar domains. Although multiple studies have probed the functions of the β clamp, a detailed understanding of the conformational dynamics of the β clamp in solution is lacking. Here we used hydrogen exchange mass spectrometry to characterize the conformation and dynamics of the intact dimer β clamp and a variant form (I272A/L273A) with a weakened ability to dimerize in solution. Our data indicate that the β clamp is not a static closed ring but rather is dynamic in solution. The three domains exhibited different dynamics, though they share a highly similar tertiary structure. Domain I, which controls the opening of the clamp by dissociating from domain III, contained several highly flexible peptides that underwent partial cooperative unfolding (EX1 kinetics) with a half-life of ~4 h. The comparison between the β monomer variant and the wild-type β clamp showed that the β monomer was more dynamic. In the monomer, partial unfolding was much faster and additional regions of domain III also underwent partial unfolding with a half-life of ~1 h. Our results suggest that the δ subunit of the clamp loader may function as a "ring holder" to stabilize the transient opening of the β clamp, rather than as a "ring opener".



Structure and Structure/Function


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