Sc. Alley et al., Building a replisome solution structure by elucidation of protein-protein interactions in the bacteriophage T4 DNA polymerise holoenzyme, J BIOL CHEM, 276(42), 2001, pp. 39340-39349
Assembly of DNA replication systems requires the coordinated actions of man
y proteins. The multiprotein complexes formed as intermediates on the pathw
ay to the final DNA polymerase holoenzyme have been shown to have distinct
structures relative to the ground-state structures of the individual protei
ns. By using a variety of solution-phase techniques, we have elucidated add
itional information about the solution structure of the bacteriophage T4 ho
loenzyme. Photocross-linking and mass spectrometry were used to demonstrate
interactions between I107C of the sliding clamp and the DNA polymerase. Fl
uorescence resonance energy transfer, analytical ultracentrifugation, and i
sothermal titration calorimetry measurements were used to demonstrate that
the C terminus of the DNA polymerase can interact at two distinct locations
on the sliding clamp. Both of these binding modes may be used during holoe
nzyme assembly, but only one of these binding modes is found in the final h
oloenzyme. Present and previous solution interaction data were used to buil
d a model of the holoenzyme that is consistent with these data.