In Escherichia coli, the DNA polymerase III (Pol III) holoenzyme, which consists of ten proteins, accounts for most of the DNA replication activity in the cell. Biochemical data has shown that in the presence of the DNA Pol III subcomponent, the β sliding clamp, replication progresses at a rate of &gt;50 kb per association event, relative to 10 nucleotides per event in the absence of the sliding clamp. Thus, β is essential for efficient and highly processive DNA replication. Crystal structures of the β sliding clamp protein confirms that it is composed of two crescent-shaped units, or protomers, with each unit made up of three domains. The two protomers combine in a head-to- tail orientation to form a homodimer ring structure. A second multiprotein subunit of DNA Pol III, the clamp loading complex δδ’γ<sub>3</sub>, is needed to load the β sliding clamp ring onto the double-stranded DNA. 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 opens the β clamp by dissociating Domain I from Domain III and then it loads 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. 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. More recently, we have made mutations at the dimer interface of the β sliding clamp that in some cases do not interfere with its biochemical or cellular functions. Currently, we are designing second generation mutations to further characterize the exact role clamp dynamics and the clamp loader complex play in opening the β ring structure and loading it onto double-stranded DNA.

Probing the conformational dynamics of the Beta sliding clamp in <i>Escherichia coli</i>

256th National Meeting (2018)

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Probing the conformational dynamics of the Beta sliding clamp in Escherichia coli