DNAX COMPLEX OF ESCHERICHIA-COLI DNA-POLYMERASE-III HOLOENZYME - THE (CHI-CENTER-DOT-PSI) COMPLEX FUNCTIONS BY INCREASING THE AFFINITY OF (TAU) AND (GAMMA) FOR DELTA-CENTER-DOT-DELTA TO A PHYSIOLOGICALLY RELEVANT RANGE

An artificial operon that contains tandem holC-holD genes was used to overproduce a complex of the chi and psi subunits of the DNA polymeras e III holoenzyme. Normally insoluble by itself, psi forms a tight solu ble complex with chi. A purification procedure that yields pure, activ e chi .psi complex in 100-mg quantities suitable for biophysical studi es is reported. Sedimentation equilibrium studies demonstrate that chi .psi is a 1:1 heterodimer. The presence of chi .psi dramatically lowe rs the level of delta .delta' required to reconstitute holoenzyme to l evels expected in vivo. That chi .psi accomplishes this by binding to gamma or tau and increasing their affinity for delta .psi' was demonst rated by surface plasmon resonance using a Pharmacia BIA-core(TM) inst rument. In the absence of delta .delta', chi .psi binds to core(TM) in strument. In the absence of delta .delta, chi .psi binds to either the gamma or tau DnaX protein with K-d = 2 nM.