title: Clamp subunit dissociation dictates bacteriophage T4 DNA polymerase holoenzyme disassembly.
abstract: 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.
authors.affiliation: Department of Chemistry, Pennsylvania State University, University Park 16802, USA.
authors.forename: P
authors.initials: P
authors.lastname: Soumillion
authors.forename: D J
authors.initials: DJ
authors.lastname: Sexton
authors.forename: S J
authors.initials: SJ
authors.lastname: Benkovic
mesh_terms: 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*
chemical_list: 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