Building a replisome solution structure by elucidation of protein-protein interactions in the bacteriophage T4 DNA polymerase holoenzyme.
Assembly of DNA replication systems requires the coordinated actions of many proteins. The multiprotein complexes formed as intermediates on the pathway to the final DNA polymerase holoenzyme have been shown to have distinct structures relative to the ground-state structures of the individual proteins. By using a variety of solution-phase techniques, we have elucidated additional information about the solution structure of the bacteriophage T4 holoenzyme. Photocross-linking and mass spectrometry were used to demonstrate interactions between I107C of the sliding clamp and the DNA polymerase. Fluorescence resonance energy transfer, analytical ultracentrifugation, and isothermal 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 holoenzyme assembly, but only one of these binding modes is found in the final holoenzyme. Present and previous solution interaction data were used to build a model of the holoenzyme that is consistent with these data.
Alley
S C
SC
Department of Chemistry, the Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Trakselis
M A
MA
Mayer
M U
MU
Ishmael
F T
FT
Jones
A D
AD
Benkovic
S J
SJ
D001710:Biotin / Q000378:metabolism; D002151:Calorimetry; D002851:Chromatography, High Pressure Liquid; D018360:Crystallography, X-Ray; D004247:DNA / Q000378:metabolism; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D020035:Holoenzymes / Q000737:chemistry*; D008163:Luminescent Measurements; D013058:Mass Spectrometry; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D014461:Ultracentrifugation; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism*
D020035:Holoenzymes; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; D001710:Biotin; D004247:DNA; D004259:DNA-Directed DNA Polymerase