Clamp subunit dissociation dictates bacteriophage T4 DNA polymerase holoenzyme disassembly.
Clamp proteins confer processivity to the DNA polymerase during DNA replication. These oligomeric proteins are loaded onto DNA by clamp loader protein complexes in an ATP-dependent manner. The mechanism by which the trimeric bacteriophage T4 clamp protein (the 45 protein) loads and dissociates from DNA was investigated as a function of its intersubunit protein-protein interactions. These interactions were continuously monitored using a fluorescence resonance energy transfer (FRET) based assay. A cysteine mutant of the 45 protein was constructed to facilitate site-specific incorporation of a fluorescent probe at the subunit interface. This site was chosen such that FRET was observed between the introduced fluorescent probe and a tryptophan residue located on the opposing subunit. By use of this fluorescently labeled 45 protein, it was possible to obtain an estimate of an apparent trimer dissociation constant from either a cooperative (0.08 +/- 0.04 microM2 at 25 degrees C) or a noncooperative (0.51 microM and 0.17 microM at 25 degrees C) model. Upon mixing the fluorescently labeled 45 protein with a 45 protein containing 4-fluorotryptophan, a nonfluorescent tryptophan analogue, subunit exchange between the two variants of the 45 protein was observed according to a reduction in intersubunit FRET. Subunit exchange rate constants measured in the presence or absence of the clamp loader (44/62 complex), the polymerase (43 protein), and/or a primer template DNA substrate demonstrate (a) that the 45 protein is not loaded onto DNA by subunit exchange and (b) that the disassembly dissociation of a stalled holoenzyme from DNA is dictated by 45 protein subunit dissociation.
Soumillion
P
P
Department of Chemistry, Pennsylvania State University, University Park 16802, USA.
Sexton
D J
DJ
Benkovic
S J
SJ
D017122:Bacteriophage T4 / Q000201:enzymology*; D003067:Coenzymes / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009282:Naphthalenesulfonates; D013050:Spectrometry, Fluorescence; D015534:Trans-Activators / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan / Q000031:analogs & derivatives; D014633:Valine / Q000235:genetics; D014764:Viral Proteins / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*
D003067:Coenzymes; D009282:Naphthalenesulfonates; D015534:Trans-Activators; D014764:Viral Proteins; C074555:gene 45 protein, Enterobacteria phage T4; C039390:4-fluorotryptophan; C006421:1,5-I-AEDANS; D014364:Tryptophan; D004259:DNA-Directed DNA Polymerase; D014633:Valine; D003545:Cysteine