Structural analysis of bacteriophage T4 DNA replication: a review in the Virology Journal series on bacteriophage T4 and its relatives

Abstract

The bacteriophage T4 encodes 10 proteins, known collectively as the replisome, that are responsible for the replication of the phage genome. The replisomal proteins can be subdivided into three activities; the replicase, responsible for duplicating DNA, the primosomal proteins, responsible for unwinding and Okazaki fragment initiation, and the Okazaki repair proteins. The replicase includes the gp43 DNA polymerase, the gp45 processivity clamp, the gp44/62 clamp loader complex, and the gp32 single-stranded DNA binding protein. The primosomal proteins include the gp41 hexameric helicase, the gp61 primase, and the gp59 helicase loading protein. The RNaseH, a 5' to 3' exonuclease and T4 DNA ligase comprise the activities necessary for Okazaki repair. The T4 provides a model system for DNA replication. As a consequence, significant effort has been put forth to solve the crystallographic structures of these replisomal proteins. In this review, we discuss the structures that are available and provide comparison to related proteins when the T4 structures are unavailable. Three of the ten full-length T4 replisomal proteins have been determined; the gp59 helicase loading protein, the RNase H, and the gp45 processivity clamp. The core of T4 gp32 and two proteins from the T4 related phage RB69, the gp43 polymerase and the gp45 clamp are also solved. The T4 gp44/62 clamp loader has not been crystallized but a comparison to the E. coli gamma complex is provided. The structures of T4 gp41 helicase, gp61 primase, and T4 DNA ligase are unknown, structures from bacteriophage T7 proteins are discussed instead. To better understand the functionality of T4 DNA replication, in depth structural analysis will require complexes between proteins and DNA substrates. A DNA primer template bound by gp43 polymerase, a fork DNA substrate bound by RNase H, gp43 polymerase bound to gp32 protein, and RNase H bound to gp32 have been crystallographically determined. The preparation and crystallization of complexes is a significant challenge. We discuss alternate approaches, such as small angle X-ray and neutron scattering to generate molecular envelopes for modeling macromolecular assemblies.

Figure 1

A cartoon model of leading and lagging strand DNA synthesis by the Bacteriophage T4 Replisome. The replicase proteins include the gp43 DNA polymerase, responsible for leading and lagging strand synthesis, the gp45 clamp, the ring shaped processivity factor involved in polymerase fidelity, and gp44/62 clamp loader, an AAA + ATPase responsible for opening gp45 for placement and removal on duplex DNA. The primosomal proteins include the gp41 helicase, a hexameric 5' to 3' ATP dependent DNA helicase, the gp61 primase, a DNA dependent RNA polymerase responsible for synthesis of primers for lagging strand synthesis, the gp32 single stranded DNA binding protein, responsible for protection of single stranded DNA created by gp41 helicase activity, and the gp59 helicase loading protein, responsible for the loading of gp41 helicase onto gp32 protected ssDNA. Repair of Okazaki fragments is accomplished by the RNase H, a 5' to 3' exonuclease, and gp30 ligase, the ATP dependent DNA ligase. Leading and lagging strand synthesis is coordinated by the replisome. Lagging strand primer extension and helicase progression lead to the formation of a loop of DNA extending from the replisome as proposed in the "trombone" model [21].

Figure 2

The molecular models, rendered to scale, of a DNA replication fork. Structures of four of ten T4 proteins are known; the RNase H (tan), the gp59 helicase loading protein (rose), the gp45 clamp (magenta), and the gp32 ssb (orange). Two additional structures from RB69, a T4 related phage, have also been completed; the RB69 gp43 polymerase (light blue) and the gp45 clamp (not shown). The E. coli clamp loader (γ complex) (pink) is used here in place of the T4 gp44/62 clamp loader, and two proteins from bacteriophage T7, T7 ligase (green) and T7 gene 4 helicase-primase (blue/salmon) are used instead of T4 ligase, and gp41/gp61, respectively.

Figure 3

The gp43 DNA polymerase from bacteriophage RB69 has been solved in complex with a DNA primer/template. The gp45 clamp from RB69 has been solved in complex with a synthetic peptide containing the PIP box motif. A.) The RB69 gp43 polymerase in complex with DNA is docked to the RB69 gp45 clamp with the duplex DNA aligned with the central opening of gp45 (gray). The N-terminal domain (tan), the 3' - 5' editing exonuclease (salmon), the palm domain (pink), the fingers domain (light blue), and thumb domain (green comprise the DNA polymerase. The C-terminal residues extending from the thumb domain contain the PCNA interacting protein box motif (PIP box) shown docked to the 45 clamp. B.) The active site of the gp43 polymerase displays the template base to the active site with the incoming dNTP base paired and aligned for polymerization. C.) The C-terminal PIP box peptide (green) is bound to a subunit of the RB69 gp45 clamp (gray).

Figure 4

Structures of T4 gp45 clamp and the E. coli clamp loader, a protein comparable to T4 gp44/62 complex. A.) The three subunits of the gp45 clamp form a ring with the large opening lined with basic residues which interact with duplex DNA. The binding pocket for interacting with PIP box peptides is shown in yellow. B.) The E. coli γ complex is shown with the γ₃ subunits (yellow, green, and cyan), the δ' stator subunit (red), and the δ wrench subunit (blue). Also indicated are the regions of the E. coli γ complex which interact with the E. coli β clamp (orange) and the P-loop motifs for ATP binding (magenta).

Figure 5

The T4 primosome is composed of the gp41 hexameric helicase, the gp59 helicase loading protein, the gp61 primase, and the gp32 single stranded DNA binding protein. A.) the gp32 single-stranded DNA binding protein binds to regions of displaced DNA near the replication fork. B.) the bacteriophage T7 gene 4 helicase domain is representative of the hexameric helicases like the T4 gp41 helicase. ATP binding occurs at the interface between domains. C.) the gp59 helicase loading protein recognizes branched DNA substrates and displaces gp32 protein from the lagging strand region adjacent to the fork. Forks of this type are generated by strand invasion during T4 recombination dependent DNA replication. D.) The two domain ATP dependent bacteriophage T7 DNA ligase represents the minimal construct for ligase activity.

Figure 6

Lagging strand DNA synthesis requires repair of the Okazaki fragments. A.) The T4 RNase H, shown with two hydrated magnesium ions (green) in the active site, is a member of the rad2/FEN-1 family of 5' - 3' exonucleases. The enzyme is responsible for the removal of lagging strand RNA primers and several bases of DNA adjacent to the RNA primer which are synthesized with low fidelity by the gp43 DNA polymerase. B.) The T4 DNA ligase, shown with ATP bound in the active site, repairs nicks present after primer removal and gap synthesis by the DNA polymerase. C.) The T4 RNase H structure has been solved with a pseudo-Y junction DNA substrate. D.) The gp32 single stranded binding protein increases the processivity of the RNase H. The two proteins interact between the C-terminal domain of RNase H and the core domain of gp32 on the 3' arm of the replication fork.