The DnaX-binding subunits δ' and ψ are bound to γ and not τ in the DNA polymerase III holoenzyme

Abstract

The DnaX complex subassembly of the DNA polymerase III holoenzyme is comprised of the DnaX proteins τ and γ and the auxiliary subunits δ, δ', χ, and ψ, which together load the β processivity factor onto primed DNA in an ATP-dependent reaction. δ' and ψ bind directly to DnaX whereas δ and χ bind to δ' and ψ, respectively (Onrust, R., Finkelstein, J., Naktinis, V., Turner, J., Fang, L., and O'Donnell, M. (1995) J. Biol. Chem. 270, 13348- 13357). Until now, it has been unclear which DnaX protein, τ or γ, in holoenzyme binds the auxiliary subunits δ, δ', χ, and ψ. Treatment of purified holoenzyme with the homobifunctional cross-linker bis(sulfosuccinimidyl)suberate produces covalently cross-linked γ-δ' and γ-ψ complexes identified by Western blot analysis. Immunodetection of cross-linked species with anti-δ' and anti-ψ antibodies revealed that no τ-δ' or τ-ψ cross-links had formed, suggesting that the δ' and ψ subunits reside only on γ within holoenzyme.