Escherichia coli DNA polymerase III holoenzyme subunits α, β, and γ directly contact the primer-template

Abstract

Escherichia coli DNA polymerase III holoenzyme forms a stable initiation complex with RNA-primed template in the presence of ATP. To determine the linear arrangement of the holoenzyme subunits along the primer-template duplex region, we cross-linked holoenzyme to a series of photo-reactive primers. Site-specific photo-cross-linking revealed that the α, β, and γ subunits formed ATP-dependent contacts with the primer-template. The α polymerase catalytic subunit covalently attached to nucleotide positions -3, -9, and -13 upstream of the primer terminus, with the most efficient adduct formation occurring at position -9. The γ subunit contacted the primer at positions -13, -18, and -22, with the strongest γ-primer interactions occurring at position -18. The β subunit predominated in cross-linking at position -22. Thus, within the initiation complex, α contacts roughly the first 13 nucleotides upstream of the 3'-primer terminus followed by γ at - 18 and β at -22, and the γ subunit remains a part of the initiation complex, bridging the α and β subunits. Analyses of the interaction of photo-activatible primer-templates with the preinitiation complex proteins (γ-complex (γ-σ-σ'-χ-Ψ) and β subunit) revealed the γ subunit within the preinitiation complex covalently attached to primer at position -3. However, addition of core DNA polymerase III to preinitiation complex, fully reconstituting holoenzyme resulted in replacement of γ by α at the primer terminus. These data indicate that assembly of holoenzyme onto a primer- template can occur in distinct stages and results in a structural rearrangement during initiation complex formation.