A single subunit directs the assembly of the Escherichia coli DNA sliding clamp loader.
Multi-protein clamp loader complexes are required to load sliding clamps onto DNA. In Escherichia coli the clamp loader contains three DnaX (tau/gamma) proteins, delta, and delta', which together form an asymmetric pentameric ring that also interacts with psichi. Here we used mass spectrometry to examine the assembly and dynamics of the clamp loader complex. We find that gamma exists exclusively as a stable homotetramer, while tau is in a monomer-dimer-trimer-tetramer equilibrium. delta' plays a direct role in the assembly as a tau/gamma oligomer breaker, thereby facilitating incorporation of lower oligomers. With delta', both delta and psichi stabilize the trimeric form of DnaX, thus completing the assembly. When tau and gamma are present simultaneously, mimicking the situation in vivo, subunit exchange between tau and gamma tetramers occurs rapidly to form heterocomplexes but is retarded when deltadelta' is present. The implications for intracellular assembly of the DNA polymerase III holoenzyme are discussed.
Park
Ah Young
AY
Department of Chemistry, University of Oxford, Oxford, UK.
Jergic
Slobodan
S
Politis
Argyris
A
Ruotolo
Brandon T
BT
Hirshberg
Daniel
D
Jessop
Linda L
LL
Beck
Jennifer L
JL
Barsky
Daniel
D
O'Donnell
Mike
M
Dixon
Nicholas E
NE
Robinson
Carol V
CV
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D004258:DNA Polymerase III / Q000737:chemistry / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D008954:Models, Biological; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; C103076:DnaX protein, Bacteria; D029968:Escherichia coli Proteins; D021122:Protein Subunits; D004258:DNA Polymerase III