Understanding DNA replication by the bacteriophage T4 replisome

Abstract

The T4 replisome has provided a unique opportunity to investigate the intricacies of DNA replication. We present a comprehensive review of this system focusing on the following: its 8-protein composition, their individual and synergistic activities, and assembly in vitro and in vivo into a replisome capable of coordinated leading/lagging strand DNA synthesis. We conclude with a brief comparison with other replisomes with emphasis on how coordinated DNA replication is achieved.

Keywords: DNA helicase; DNA polymerase; DNA primase; DNA replication; DNA-binding protein; bacteriophage.

Figure 1.

Kinetic scheme for the T4 polymerase. The minimal kinetic scheme for the polymerase and exonuclease activities of the T4 gp43 enzyme as integrated in an idling process, i.e. the nucleotide is inserted and then excised, is shown. Reprinted with permission from ACS Publications, obtained via RightsLink Copyright Clearance Center (Capson, T. L., Peliska, J. A., Kaboord, B. F., Frey, M. W., Lively, C., Dahlberg, M., and Benkovic, S. J. (1992) Kinetic characterization of the polymerase and exonuclease activities of the gene 43 protein of bacteriophage T4. Biochemistry 31, 10984–10994).

Figure 2.

Representation of the bacteriophage T4 DNA polymerase holoenzyme assembly process derived from pre- and steady-state kinetics. A proposed structural representation of the key steps 1, 3, 4, 6, 8, and 10 is depicted. Orange boxes highlight the important conclusions, namely the hydrolysis of ATP in step 3 to open the clamp, the hydrolysis of ATP in step 6 to close the clamp around DNA, and the dissociation of gp44/62 from the holoenzyme in step 10. Figure has been adapted with permission from Elsevier, obtained via RightsLink by Copyright Clearance Center (Trakselis, M. A., Berdis, A. J., and Benkovic, S. J. (2003) Examination of the role of the clamp-loader and ATP hydrolysis in the formation of the bacteriophage T4 polymerase holoenzyme. J. Mol. Biol. 326, 435–451).

Figure 3.

Assembly mechanism of the T4 lagging-strand primosome on forked DNA. The gp32 protein binds to forked DNA with either subsequent or concurrent binding of gp59. Subsequently, gp41 binds to gp59 and is loaded onto the forked DNA in the presence of nucleotide. ATP hydrolysis is required for gp41 to displace gp32 and gp59, either directly or by translocation. The gp61 protein then binds and interacts closely with gp41 on forked DNA. In the absence of gp32 and gp59, both gp41 and gp61 bind to forked DNA. Figure has been adapted to reflect the current opinion on primase stoichiometry with permission from the National Academy of Sciences (© (2005) National Academy of Sciences. Zhang, Z., Spiering, M. M., Trakselis, M. A., Ishmael, F. T., Xi, J., Benkovic, S. J., and Hammes, G. G. (2005) Assembly of the bacteriophage T4 primosome: single-molecule and ensemble studies. Proc. Natl. Acad. Sci. U.S.A. 102, 3254–3259).

Figure 4.

Proposed model for pppRNA·primase complex as the signal for lagging-strand initiation. A, during the replication phase of Okazaki fragment synthesis, primase subunits within a replisome stochastically synthesize 5-mer RNA primers at priming sites along the lagging-strand DNA; these primers are stabilized on the DNA by primase subunits dissociated from the primosome. B, pppRNA·primase complex forms a block, triggering recycling of the lagging-strand polymerase in the signaling model, and results in a gap between Okazaki fragments. C, collision with the previous Okazaki fragment triggers recycling of the lagging-strand polymerase in the collision model and results in no gap between Okazaki fragments. D, holoenzyme is assembled on the new RNA primer to initiate the next Okazaki fragment. Reprinted with permission from National Academy of Sciences (© (2005) National Academy of Sciences. Spiering, M. M., Hanoian, P., Gannavaram, S., and Benkovic, S. J. (2017) RNA primer–primase complexes serve as the signal for polymerase recycling and Okazaki fragment initiation in T4 phage DNA replication. Proc. Natl. Acad. Sci. U.S.A. 114, 5635–5640).