CLAMP SUBUNIT DISSOCIATION DICTATES BACTERIOPHAGE-T4 DNA-POLYMERASE HOLOENZYME DISASSEMBLY

Clamp proteins confer processivity to the DNA polymerase during DNA re plication. These oligomeric proteins are loaded onto DNA by clamp load er protein complexes in an ATP-dependent manner. The mechanism by whic h the trimeric bacteriophage T4 clamp protein (the 45 protein) loads a nd dissociates from DNA was investigated as a function of its intersub unit protein-protein interactions. These interactions were continuousl y monitored using a fluorescence resonance energy transfer (FRET) base d assay. A cysteine mutant of the 45 protein was constructed to facili tate site-specific incorporation of a fluorescent probe at the subunit interface. This site was chosen such that FRET was observed between t he introduced fluorescent probe and a tryptophan residue located on th e opposing subunit. By use of this fluorescently labeled 45 protein, i t was possible to obtain an estimate of an apparent trimer dissociatio n constant from either a cooperative (0.08 +/- 0.04 mu M-2 at 25 degre es C) or a noncooperative (0.51 mu M and 0.17 mu M at 25 degrees C) mo del. Upon mixing the fluorescently labeled 45 protein with a 45 protei n containing 4-fluorotryptophan, a nonfluorescent tryptophan analogue, subunit exchange between the two variants of the 45 protein was obser ved according to a reduction in intersubunit FRET. Subunit exchange ra te 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 D NA by subunit exchange and (b) that the disassembly dissociation of a stalled holoenzyme from DNA is dictated by 45 protein subunit dissocia tion.