Examination of the role of the clamp-loader and ATP hydrolysis in the formation of the bacteriophage T4 polymerase holoenzyme.
Transient kinetic analyses further support the role of the clamp-loader in bacteriophage T4 as a catalyst which loads the clamp onto DNA through the sequential hydrolysis of two molecules of ATP before and after addition of DNA. Additional rapid-quench and pulse-chase experiments have documented this stoichiometry. The events of ATP hydrolysis have been related to the opening/closing of the clamp protein through fluorescence resonance energy transfer (FRET). In the absence of a hydrolysable form of ATP, the distance across the subunit interface of the clamp does not increase as measured by intramolecular FRET, suggesting gp45 cannot be loaded onto DNA. Therefore, ATP hydrolysis by the clamp-loader appears to open the clamp wide enough to encircle DNA easily. Two additional molecules of ATP then are hydrolyzed to close the clamp onto DNA. The presence of an intermolecular FRET signal indicated that the dissociation of the clamp-loader from this complex occurred after guiding the polymerase onto the correct face of the clamp bound to DNA. The final holoenzyme complex consists of the clamp, DNA, and the polymerase. Although this sequential assembly mechanism can be generally applied to most other replication systems studied to date, the specifics of ATP utilization seem to vary across replication systems.
Trakselis
Michael A
MA
Department of Chemistry, 415 Wartik Laboratory, The Pennsylvania State University, University Park, PA 16802, USA.
Berdis
Anthony J
AJ
Benkovic
Stephen J
SJ
D000255:Adenosine Triphosphate / Q000378:metabolism*; D017122:Bacteriophage T4 / Q000201:enzymology*; D004269:DNA, Bacterial / Q000737:chemistry* / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D020035:Holoenzymes; D006868:Hydrolysis; D008715:Methionine / Q000737:chemistry / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013460:Sulfur Isotopes; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism
D004269:DNA, Bacterial; D020035:Holoenzymes; D013460:Sulfur Isotopes; D015534:Trans-Activators; D014764:Viral Proteins; C074855:gene 44 protein, Enterobacteria phage T4; C074555:gene 45 protein, Enterobacteria phage T4; C088659:gp62 protein, bacteriophage T4; D000255:Adenosine Triphosphate; D008715:Methionine; D004259:DNA-Directed DNA Polymerase