Proteolysis of the proofreading subunit controls the assembly of Escherichia coli DNA polymerase III catalytic core.
The C-terminal region of the proofreading subunit (epsilon) of Escherichia coli DNA polymerase III is shown here to be labile and to contain the residues (identified between F187 and R213) responsible for association with the polymerase subunit (alpha). We also identify two alpha-helices of the polymerase subunit (comprising the residues E311-M335 and G339-D353, respectively) as the determinants of binding to epsilon. The C-terminal region of epsilon is degraded by the ClpP protease assisted by the GroL molecular chaperone, while other factors control the overall concentration in vivo of epsilon. Among these factors, the chaperone DnaK is of primary importance for preserving the integrity of epsilon. Remarkably, inactivation of DnaK confers to Escherichia coli inviable phenotype at 42 degrees C, and viability can be restored over-expressing epsilon. Altogether, our observations indicate that the association between epsilon and alpha subunits of DNA polymerase III depends on small portions of both proteins, the association of which is controlled by proteolysis of epsilon. Accordingly, the factors catalysing (ClpP, GroL) or preventing (DnaK) this proteolysis exert a crucial checkpoint of the assembly of Escherichia coli DNA polymerase III core.
Bressanin
Daniela
D
Department of Industrial Chemistry, University of Bologna, Viale Risorgimento 4, 40136 Bologna, Italy.
Stefan
Alessandra
A
Piaz
Fabrizio Dal
FD
Cianchetta
Stefano
S
Reggiani
Luca
L
Hochkoeppler
Alejandro
A
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D004258:DNA Polymerase III / Q000737:chemistry / Q000378:metabolism*; D049071:Endopeptidase Clp / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis
D029968:Escherichia coli Proteins; C074904:DNA polymerase III, alpha subunit; D004258:DNA Polymerase III; C483572:ClpP protease, E coli; D049071:Endopeptidase Clp