Replisome Architecture and Dynamics in Escherichia coli

Abstract

The field of DNA replication was launched upon discovery of the first DNA polymerase by Kornberg and Lehman (1,2). DNA polymerase I displayed the novel and highly exciting property of template-directed enzymatic action, and like a split personality, DNA polymerase I could also degrade DNA, from either direction. Escherichia coli is now known to contain five different DNA polymerases. The chromosomal replicase is DNA polymerase III, while the damage-inducible DNA polymerases II, IV, and V play roles in DNA repair. DNA polymerase I is involved in both replication and repair and remains the most advanced model for the structure and function of DNA polymerase action.DNA polymerase III (pol III) 2 functions in the context of a multiprotein apparatus called DNA pol III holoenzyme (reviewed in (3-5)). pol III holoenzyme contains 10 different proteins, which assort into three major functional units: 1) pol III core, 2) the ␤ sliding clamp, and 3) the ␥/ complex clamp loader. The holoenzyme contains two copies of the pol III core, which are connected by the attachment to one clamp loader. The holoenzyme functions within the context of a dynamic replisome containing pol III holoenzyme, a hexameric DnaB helicase, DnaG primase, and SSB. During replisome function, contacts between these proteins are in a constant state of change. This review briefly summarizes the architecture and dynamic behavior of the E. coli replisome. The Clamp and Clamp LoaderThe ␤ sliding clamp is a homodimer in the shape of a ring, which encircles DNA (Fig. 1A). The ␤ clamp slides on DNA and binds the pol III core, thereby acting as a mobile tether and converting the normally distributive pol III core into a highly processive and rapid polymerase capable of incorporating 500 -1,000 nucleotides per second (Fig. 1C). The crystal structure of the ␤ dimer reveals a 6-fold pseudo symmetry that arises from a domain that is repeated three times in the monomer giving the dimer a 6-fold appearance (6). The eukaryotic PCNA clamp and phage T4 gp45 clamp are also six-domain rings, but the monomeric unit contains only two domains and trimerizes to form a six-domain ring (7).Clamps do not self-assemble onto DNA but require a multiprotein clamp loader, which harnesses the energy of ATP hydrolysis to open and close the clamp around DNA (Fig. 1B). The E. coli ␥ complex clamp loader contains five subunits that are essential for clamp loading activity, three ␥ protomers and one copy each of ␦ and ␦Ј. The small and subunits are not required for clamp loading, but they stabilize the clamp loader and stimulate its activity at elevated ionic strength.The ␥, ␦, and ␦Ј clamp loading subunits are members of the AAAϩ family (ATPases associated with a variety of cellular functions) (8). The subunits consist of three domains, and a AAAϩ region of homology is localized to the first two domains (9). Many AAAϩ proteins are homohexamers arranged in a symmetric circle (10, 11). The ␥ 3 ␦␦Ј pentamer forms an asymmetric circle, and there is a gap in place of the "missing sixth subunit"...

Acidic C-terminal tail of the ssDNA-binding protein of bacteriophage T7 and ssDNA compete for the same binding surface

“…Once assigned to such mundane roles as eliminating secondary structure in ssDNA and protecting DNA from cleavage by nucleases, they are now emerging as key components in coordinating reactions at replication forks (2,3).…”

Heterohexamer of 56- and 63-kDa Gene 4 Helicase-Primase of Bacteriophage T7 in DNA Replication

“…In the replisome of E. coli, three DnaG primases are associated with a hexamer of DnaB helicase to form a functional complex (1,13). The crystal structures have identified three DnaG primases bound to the hexameric DnaB helicase (45).…”

“…In other prokaryotic systems such as Escherichia coli and bacteriophage T4, separate genes encode the two proteins. Nonetheless, the primase and helicase in the other systems have interacting domains, and the two proteins function together at the replication fork (1,12,13). Rather than having interacting motifs at the C terminus of the primase and the N terminus of the helicase, the two domains in gp4 are covalently connected via a linker of 26 residues (residues 246 -271).…”

The bacteriophage P1 hot gene, encoding a homolog of the E. coli DNA polymerase III θ subunit, is expressed during both lysogenic and lytic growth stages

“…The bacteriophage P1 hot gene encodes a homolog of the θ subunit of E. coli DNA polymerase III holoenzyme, which is responsible for replicating the bacterial chromosome (for reviews, see [1][2][3]). Specifically, θ is a component of the pol III core, which consists of the α subunit (DNA polymerase), the ε subunit (3′ exonuclease that serves as proofreader for Pol III insertion errors), and θ, which are tightly bound together in the linear order α-ε-θ.…”