Sliding Clamp of the Bacteriophage T4 Polymerase Has Open and Closed Subunit Interfaces in Solution

Abstract

The sliding clamps of bacteriophage T4 (gp45), Escherichia coli (beta clamp), and yeast (PCNA) are required for processive DNA synthesis by their cognate DNA polymerases. The X-ray crystal structures of all three of these clamps have been shown to be closed, circular complexes. This paper reports investigations of the solution structure of bacteriophage T4 gp45 by analytical ultracentrifugation, fluorescence, and hydrodynamic modeling. Mutants of gp45 with inter- and intrasubunit disulfide bonds were created to alter the solution structure of gp45, with additional mutagenesis used to investigate the importance of the proline-rich loop region found between the two domains of each gp45 monomer. The wild-type gp45 trimer assembles from monomers cooperatively with a dissociation constant of 0.21 microM2 and values between 0.088 and 0. 32 microM2 for the mutants. Velocity ultracentrifugation experiments showed that wild-type gp45 possesses a sedimentation coefficient strongly dependent on concentration, typical of asymmetric or elongated molecules, that when extrapolated to zero concentration yields a sedimentation coefficient of 4.0 S. The loop and the disulfide mutants exhibited sedimentation coefficients with little concentration dependence, typical of symmetric or spherical molecules, that when extrapolated to zero concentration yielded sedimentation coefficients of 4.4-4.8 S. The lower sedimentation coefficient in the former case is consistent with wild-type gp45 being more asymmetric or elongated than the mutant forms. Fluorescence-resonance energy-transfer experiments were used to measure the distance between two amino acids (W91 and V162C-coumarin) on opposite sides of the gp45 subunit interface. For an intrasubunit disulfide mutant, the distance between these two amino acids was determined to be 19 A (14 A in the X-ray crystal structure), consistent with a closed complex. For the mutants without intrasubunit disulfides, the efficiency of fluorescence-resonance energy transfer was in accord with a model of gp45 being an open complex composed of two closed subunit interfaces and a third open interface separated by a distance of 35-38 A. The collective data supplemented with hydrodynamic modeling were consistent with gp45 subunit separation achieved within the plane of the gp45 ring.

Creating a dynamic picture of the sliding clamp during T4 DNA polymerase holoenzyme assembly by using fluorescence resonance energy transfer

“…Oligonucleotide primers and substrates were prepared as previously described (14,33,34). The forked bio62͞34͞36mer primer-template blocked with streptavidin (34) was used as the DNA substrate in all experiments.…”

“…pET26b-W92F͞S158C͞W199F and pET26b-W92F͞ T168C͞W199F were then created from the respective mutants above by using fragments AH and BG in the overlap extension reaction yielding a new BsiEI site. Purification and labeling with CPM were performed as described previously (12,33). Mutations were confirmed by DNA sequencing.…”

“…Initially, we discovered that one of the three subunit interfaces of gp45 is open to a distance of 35-38 Å, and the other two interfaces are closed with a distance of 19 Å (as measured between W91 on one subunit interface and a fluorophore on the opposite interface) (33). By creating three site-specific mutants of gp45 (V163C, S158C, and T168C) singly labeled with a fluorophore, energy transfer was detected from a single endogenous tryptophan (W91) during holoenzyme assembly.…”

The dynamic processivity of the T4 DNA polymerase during replication

“…The polymerase C-terminal region was shown to interact with the interdomain loop of one of the clamp subunits. However, solution studies revealed a clamp structure with one open and two closed interfaces (20) and strongly argued for an interaction between the polymerase C terminus and the open clamp subunit interface (21,22). The x-ray data and its associated holoenzyme model which used the weaker interdomain binding site of gp45 was predicted, however, to play some role in holoenzyme assembly, DNA replication, translesion bypass, or other replication associated processes (19,21).…”

Out-of-plane motions in open sliding clamps: Molecular dynamics simulations of eukaryotic and archaeal proliferating cell nuclear antigen

“…That the clamp bound to the clamp loader is in a closed form in the crystal structure of the RFC-PCNA complex came as a surprise, because the engagement of an ATP-bound clamp loader with the clamp is known to result in clamp opening (15,19,20). The crystal structure may correspond to a state of the system just before the release of the clamp on DNA.…”