DNA Helicase-Polymerase Coupling in Bacteriophage DNA Replication

Abstract

Bacteriophages have long been model systems to study the molecular mechanisms of DNA replication. During DNA replication, a DNA helicase and a DNA polymerase cooperatively unwind the parental DNA. By surveying recent data from three bacteriophage replication systems, we summarized the mechanistic basis of DNA replication by helicases and polymerases. Kinetic data have suggested that a polymerase or a helicase alone is a passive motor that is sensitive to the base-pairing energy of the DNA. When coupled together, the helicase-polymerase complex is able to unwind DNA actively. In bacteriophage T7, helicase and polymerase reside right at the replication fork where the parental DNA is separated into two daughter strands. The two motors pull the two daughter strands to opposite directions, while the polymerase provides a separation pin to split the fork. Although independently evolved and containing different replisome components, bacteriophage T4 replisome shares mechanistic features of Hel-Pol coupling that are similar to T7. Interestingly, in bacteriophages with a limited size of genome like Φ29, DNA polymerase itself can form a tunnel-like structure, which encircles the DNA template strand and facilitates strand displacement synthesis in the absence of a helicase. Studies on bacteriophage replication provide implications for the more complicated replication systems in bacteria, archaeal, and eukaryotic systems, as well as the RNA genome replication in RNA viruses.

Keywords: DNA replication; bacteriophage T4; bacteriophage T7; bacteriophage Φ29; helicase; polymerase.

Figure 1

Comparison of the kinetic parameters of dsDNA unwinding in T7, T4, and Φ29 systems. (a) The rate of Pols (green) translocation on the ssDNA. (b) The rate of Pols (green) translocation on the dsDNA. (c) The rate of the hexameric Hels (blue) translocation on the ssDNA. (d) The rate of the Hels (blue) translocation on the dsDNA. (e) The rate of the coupled Hels (blue)–Pols (green) action on the dsDNA. The arrows indicate the direction of Hel and Pol translocation, with their lengths correlated with the translocation processivity and their thickness correlated with the translocation speed.

Figure 2

Structure of the T7 Hel–Pol complex on a replication fork. (a) Diagram of the replication fork with the coordinated leading-strand and lagging-strand DNA synthesis. (b) The Hel–Pol–DNA fork structure. (c) A zoom-in view of the DNA fork bound by the T7 Pol and the Hel. The Pol, Hel, and primase are colored green, blue, and grey, respectively. The β-loop for the T7 Pol is shown as black cartoons and resides at the fork junction. The W579 from the β-loop stacks with the first base pair of the parental DNA. The T7 Hel DNA binding loops are shown in a blue ribbon in panel (c). The position of the charged–charge interactions between Hel and Pol are indicated by pink and blue symbols. Structures are adapted from [21].

Figure 3

DNA binding in T7 (a), RB69 (b), and Φ29 (c) Pols. The electrostatic surfaces of proteins are shown, with negatively and positively charged surfaces colored as red and blue, respectively. The dsDNA binding surface in T7 Pol and the potential dsDNA binding surfaces in T4 and Φ29 Pols are highlighted with black circles. The β-loops in the T7 Pol and the T4 Pol and the TPR2 domain in the Φ29 Pol are shown as black cartoons. An arrow indicates the TPR2 tunnel in the Φ29 Pol. Structures are adapted from [21,38,39].