(NAS Colloquium) Links Between Recombination and Replication: Vital Roles of Recombination

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Colloquium on Links Between Recombination and Replication: Vital Roles of Recombination

Colloquium

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

Michael A.Trakselis, Stephen C.Alley*, Ernesto Abel-Santos, and Stephen J.Benkovic^†

Department of Chemistry, 414 Wartik Laboratory, The Pennsylvania State University, University Park, PA 16802

The coordinated assembly of the DNA polymerase (gp43), the sliding clamp (gp45), and the clamp loader (gp44/62) to form the bacteriophage T4 DNA polymerase holoenzyme is a multistep process. A partially opened toroid-shaped gp45 is loaded around DNA by gp44 62 in an ATP-dependent manner. Gp43 binds to this complex to generate the holoenzyme in which gp45 acts to topologically link gp43 to DNA, effectively increasing the processivity of DNA replication. Stopped-flow fluorescence resonance energy transfer was used to investigate the opening and closing of the gp45 ring during holoenzyme assembly. By using two site-specific mutants of gp45 along with a previously characterized gp45 mutant, we tracked changes in distances across the gp45 subunit interface through seven conformational changes associated with holoenzyme assembly. Initially, gp45 is partially open within the plane of the ring at one of the three subunit interfaces. On addition of gp44/62 and ATP, this interface of gp45 opens further in-plane through the hydrolysis of ATP. Addition of DNA and hydrolysis of ATP close gp45 in an out-of-plane conformation. The final holoenzyme is formed by the addition of gp43, which causes gp45 to close further in plane, leaving the subunit interface open slightly. This open interface of gp45 in the final holoenzyme state is proposed to interact with the C-terminal tail of gp43, providing a point of contact between gp45 and gp43. This study further defines the dynamic process of bacteriophage T4 polymerase holoenzyme assembly.

DNA replication requires the coordinated assembly of many proteins to form a replisome responsible for copying an organism’s genome. The generation of two holoenzymes, one for leading- and one for lagging-strand synthesis, proceeds stepwise with many intermediates. The bacteriophage T4 DNA polymerase holoenzyme is derived from the polymerase (gp43), the clamp (gp45), and the clamp-loader complex (gp44/62) (1, 2). Analogous proteins are found in both the Escherichia coli holoenzyme, consisting of the DNA polymerase III, the β clamp, and the clamp-loading γ complex, and in the eukaryotic holoenzyme, consisting of DNA polymerase δ, the proliferating cell nuclear antigen (PCNA) clamp, and the clamp-loading RF-C complex (3–7). The sliding clamp, gp45, of bacteriophage T4 is a ring-shaped protein trimer (Fig. 1A) that is an essential component of the holoenzyme, which acts by conferring the property of processivity on the DNA polymerase. X-ray crystallography has shown the clamps from the various systems to be similar in overall structure but different in oligomeric structure, with gp45 (8, 9) and PCNA (10) formed as trimers and the β clamp (11) as a dimer. The clamp-loader complex, gp44/62, is a 4:1 complex of gp44 and gp62 that sequentially hydrolyzes two sets of two molecules of ATP while loading gp45 onto DNA and also facilitates the gp45-gp43 interaction (12–14). The polymerase, gp43, incorporates nucleotides in a 5′ to 3′ direction complementary to a DNA template. The coordinated actions of these proteins in bacteriophage T4 result in a highly efficient model system for studying DNA replication.

Without the crystal structure of gp44/62 or other structural information, an exact model of the interaction between gp45, gp44/62, gp43, and the holoenzyme waits to be determined. However, the structures of individual components of the holoenzyme have been elucidated as well as specific points of interaction between the proteins. The x-ray crystal structure of gp43 from bacteriophage RB69 has been solved (15) and shares 74% sequence similarity with the bacteriophage T4 gp43 (16). Only one region of gp43 from T4 lacks significant sequence homology to gp43 from RB69, and this area has been implicated in the dimerization of the polymerases (17). Photocrosslinking has determined that gp44/62 and gp43 interact with gp45 on the same “rough” face of the clamp (18–20). Recently, we have shown that the C-terminal tail of gp43 crosslinks to amino acid residues within the subunit interface of gp45 (21) and is absolutely required for holoenzyme assembly (22).

A variety of steady-state and presteady-state techniques have been used to investigate the kinetic assembly of the holoenzyme in bacteriophage T4 (23–25) and in E. coli (26–30) Both systems are assembled stepwise to form their holoenzymes. In bacteriophage T4, gp44/62 hydrolyzes two molecules of ATP to open gp45 and then two more molecules of ATP on interaction with DNA (14, 23, 24, 31). In E. coli, the γ complex hydrolyzes two to three molecules of ATP (32) on interaction with DNA. It has not been unequivocally determined whether this hydrolysis is sequential or simultaneous because of conflicting results (26, 30). In either case, the structural changes that occur from ATP hydrolysis in the clamp or clamp-loader complex have not been identified.

In the present experiments, we investigated the directionality of gp45 subunit interface opening and closing during holoenzyme assembly by using presteady-state fluorescence resonance energy transfer (FRET) techniques. 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

This paper results from the National Academy of Sciences colloquium, “Links Between Recombination and Replication: Vital Roles of Recombination,” held November 10–12, 2000, in Irvine, CA.

Abbreviations: CPM, 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin; FRET, fluorescence resonance energy transfer.

*	Present address: Chiron Corporation, 201 Elliott Avenue West, Suite 150, Seattle, WA 98119.
^†	To whom reprint requests should be addressed. E-mail: sjb1@chem.psu.edu.

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