The DNA polymerase III holoenzyme: An asymmetric dimeric replicative complex containing distinguishable leading and lagging strand polymerases

The DNA polymerase III holoenzyme is a multi-functional enzyme that possesses the ability to replicate both the leading and lagging strand of DNA. My research examines not only the spatial arrangement of protein subunits within holoenzyme, but how this arrangement coupled with holoenzyme's intrinsic enzymatic activities facilitates semi-discontinuous replication.; I demonstrated that the ability of holoenzyme to replicate SSB-coated DNA templates in the presence of elevated ionic strength arises from a direct interaction of the χψ subunits of holoenzyme and SSB. This interaction allowed a minimal polymerase assembly of Pol IIIτψχ to replicate SSB-coated DNA. These findings are consistent with χψ strengthening a holoenzyme-SSB-coated lagging strand interaction at the replication fork.; The presence of one copy of δδ′χψ within the DnaX-complex imparts a structural asymmetry to the holoenzyme. Using chemical crosslinking I demonstrated that within authentic holoenzyme, δδ ′χψ reside exclusively on γ. The association of δδ ′χψ with domain III of DnaX₄ results in a DnaX-complex with an overall stoichiometry of DnaX₃δ ₁δ′₁χ₁ψ ₁. Using DnaX proteins containing different structural domains I demonstrated that domain III of DnaX also contains the binding surface necessary for DnaX oligomerization.; Holoenzyme forms ATP-dependent initiation complexes on primed DNA. I demonstrated that the non-hydrolyzable ATP-analog, ATPγS, supported the formation of a leading strand complex which loaded and replicated the lagging strand only in the presence of ATP, β and SSB. Once formed, the dimeric replication complex disassembled in the opposite order by which it assembled. Upon ATPγS-mediated dissociation, the leading strand polymerase was refractory to disassembly allowing cycling to occur exclusively on the lagging strand. This established holoenzyme as an intrinsic asymmetric dimer with distinguishable leading and lagging strand polymerases.; I developed an assembly and purification method for the two possible forms of Pol III*: (αϵ&thetas;)₁τ₁γ ₂δδ′χψ and (αϵ&thetas;) ₂τ₂γ₁δδ′χψ. Using these complexes as standards for holoenzyme, I compared their ability to form initiation complexes in the presence of ATP and ATPγS. (αϵ&thetas;) ₂τ₂γ₁δδ′χψ and holoenzyme had identical properties as they were able to form both a leading and lagging strand complex while (αϵ&thetas;)₁τ ₁γ₂δδ′χψ could only form a leading strand complex. Based on this result we proposed that holoenzyme has the composition: (αϵ&thetas;)₂τ ₂γ₁δ₁δ′ ₁χ₁ψ₁.