DnaX Complex of Escherichia coli DNA Polymerase III Holoenzyme THE χ·ψ COMPLEX FUNCTIONS BY INCREASING THE AFFINITY OF τ AND γ FOR δ·δ ′ TO A PHYSIOLOGICALLY RELEVANT RANGE

摘要: An artificial operon that contains tandem holC-holD genes was used to overproduce a complex of the χ and ψ subunits DNA polymerase III holoenzyme. Normally insoluble by itself, forms tight soluble with χ. A purification procedure yields pure, active χ·ψ in 100-mg quantities suitable for biophysical studies is reported. Sedimentation equilibrium demonstrate 1:1 heterodimer. The presence dramatically lowers level δ·δ′ required reconstitute holoenzyme levels expected vivo. That accomplishes this binding γ or τ increasing their affinity demonstrated surface plasmon resonance using Pharmacia BIAcoreTM instrument. In absence δ·δ′, binds either DnaX protein Kd = 2 nM.