DNA Polymerase III: Running Rings around the Fork

within 30–40 min, RNA primers are generated on the Metabolic processes are often orchestrated by the coorlagging strand template every 1–2 s at average intervals dinated action of multiple protein components. Because of 1–2 kb. The elongationof each primer by pol III holoenof the complexity of such enzymatic mechanisms, the zyme takes place at a rate of about 1000 nucleotides participant proteins are aptly referred to as constituting per second and is highly processive owing to the presenzymatic “machinery.” Deciphering the inner workings ence of the sliding clamp subunit. The discontinuous of the multiprotein machines that mediate processes, mode of replication demands that pol III must cycle to such as DNA replication and transcription, is a major the next RNA primer upon completion of each Okazaki goal of biology, but is a technically demanding task fragment. This raises two potential difficulties. First, the owing to the difficulty in reassembling functional comcycling process must be very rapid, occupying only a plexes from purified components outside of the cell. fraction of the total time devoted to polymerization. Since its discovery nearly 25 years ago, the replicase Rapid cycling is essential to ensure that the synthesis of Escherichia coli, DNA polymerase III (pol III) holoenof the lagging strand keeps pace with the synthesis of zyme, has been extensively studied as a model replicathe leading strand. Second, the requirement for cycling tion machine (Kornberg and Baker, 1992; Kelman and of pol III would appear, at least at first sight, to be at O’Donnell, 1995). The 10 protein subunits of pol III holoodds with the highly processive character of the polyenzyme function in cooperation with other replication merization process. Recent experiments by O’Donnell proteins to carry out the duplication of the entire 4.4 Mb and colleagues suggest that these problems are solved E. coli chromosome in 30–40 min. Over the past decade, by a remarkable mechanism that involves the partial work in several laboratories resulted in the identification disassembly and reassembly of the holoenzyme strucof the genes encoding all 10 subunits and the high level ture during the synthesis of each Okazaki fragment (Stuexpression of the corresponding gene products. This kenberg et al., 1994; Naktinis et al., 1996 [this issue of accomplishment has made possible elegant biochemiCell]). The mechanism is powered by ATP hydrolysis cal studies that have brought understanding of thestrucand is controlled by specific protein–protein and proture and function of the pol III holoenzyme to a level of tein–DNA interactions. detail unmatched by other protein machines. Pol III holoenzyme is composed of 10 unique subunits At the E. coli replication fork, the DNA duplex is proand harbors at least three essential enzymatic activities gressively unwound by the action of a DNA helicase, (Table 1). The enzyme contains four distinct functional and the exposed single strands serve as templates for components: the core polymerase (aeu), which contains the synthesis of short RNA primers by the primase and both DNA polymerase (a) and proofreading exonuclease associated proteins. The role of pol III holoenzyme is to (e) activities; the sliding clamp (b dimer), which confers elongate newly synthesized primers to generate the two processivity by tethering the holoenzyme to the temprogeny strands. Because of the antiparallel nature of plate DNA; the clamp loader or g complex (g2d1d91x1c1), the DNA duplex, two different modes of priming are which assembles b clamps onto the DNA in an ATPrequired. Polymerization of one progeny strand (the dependent reaction; the linker protein (t2), which binds “leading” strand) occurs in the same direction as the two core polymerase molecules and one g complex. The replication fork moves. Thus, only a single priming event structure of a stable subassembly of pol III, known as is required, after which the leading strand is elongated pol III*, has been studied in detail by a variety of methcontinuously by pol III holoenzyme. Leading strand synods. Pol III* contains two core polymerases, one t dimer thesis is highly processive owing to the presence of a and one g complex (Figure 1). The enzyme exhibits “sliding clamp” subunit that tethers the polymerase to greatly reduced processivity relative to the holoenzyme the template. Polymerization of the second progeny because it lacks the b subunit, which readily dissociates strand (the “lagging” strand) occurs in the direction opfrom the holoenzyme during purification. Addition of the posite to replication fork movement. Thus, elongation b subunit to pol III* regenerates the holoenzyme and of the lagging strand is a discontinuous process involvrestores processivity. The pol III* complex can be reconing the repeated synthesis of RNA primers that are then stituted from individual subunits, and a general picture extended into short DNA chains (Okazaki fragments) by of its overall organization has been deduced from depol III holoenzyme. Completion of the lagging strand tailed analysis of subunit–subunit interactions (Onrust requires a repair system to remove the primers, fill in et al., 1995, and references therein) (Figure 1). As men-