DNA replication, the basis for biological inheritance, is a fundamental process occurring in all living organisms requiring the complete genome to be copied before each cell division. Replisome mediated DNA replication in bacteriophage T4 requires the coordinated action of eight proteins to efficiently and accurately replicate DNA on both the leading and lagging strands. This relatively simple DNA replication model system possesses functional analogs in prokaryotes and eukaryotes. It includes the leading- and lagging-strand DNA polymerases that synthesize the daughter DNA strands, and their associated clamps and clamp loader, collectively known as the holoenzyme. The replisome also contains a primosome consisting of a primase to make short RNA chains to initiate new DNA fragments on the lagging strand, a helicase to unwind the parental duplex, and a helicase loading protein. Single-stranded DNA (ssDNA)-binding protein coats the lagging strand before it is replicated.

What is the structure of the T4 replisome?

An extensive body of literature exists on the functioning of the individual T4 replication proteins, but there is little direct structural data on the architecture of an intact functioning replisome. In collaboration with Dr. Huilin Li at the Van Andel Institute, we are using cryo-EM and single-particle reconstruction methods to determine a structure of the T4 replisome.

We have solved a series of cryo-EM structures of T4 primosome assembly intermediates at resolutions up to 2.7 Å. We show that the helicase is an open spiral in the absence of ssDNA and that ssDNA binding triggers a large-scale scissor-like conformational change that drives the open spiral to a closed ring that activates the helicase (Figure 1A). We found that the activation of the gp41 helicase exposes a cryptic hydrophobic primase binding surface allowing for the recruitment of the gp61 primase. The primase binds the helicase in a bipartite mode in which the Zn-binding domain (ZBD) and the RNA polymerase domain (RPD) each contain a helicase-interacting motif that bind to separate helical hairpin dimers of the helicase ring, leading to the assembly of one primase on the helicase hexamer (Figure 1B). Based on two observed primosome conformations – one in a DNA-scanning mode and the other in a post RNA primer-synthesis mode – we suggest that the linker loop between the gp61 ZBD and RPD contributes to determining the length of the pentaribonucleotide primer synthesized by the T4 primase. Our study reveals the T4 primosome assembly process and sheds light on the RNA primer synthesis mechanism.

Figure 1. Cryo-EM study of the T4 primosome subassembly. A) Side views of the hexameric helicase undergoing conformational changes from the inactive, open-spiral structure (left; gap between subunits A and F) to the active, closed-ring structure upon ssDNA binding (right). Subunits A-D are shown as gray surfaces. The C-terminal domains of subunits E and F are shown in colored surfaces (pink and salmon) while their N-terminal domains (light and dark blue) and Linking Helixes (green) are shown in cartoon form. The N-terminal helical hairpins (enclosed in a red, dashed-line shape) close in a scissor-like motion that increases their crossing angle from 100º in the open spiral form to 165º and almost antiparallel in the closed-ring form. B) Three primase binding poses on the hexameric helicase are observed in the presence of ssDNA and ATPγS. The upper panels are sketches of the three EM maps shown in the lower panels (particle population given in parentheses). These poses are similar, but non-equivalent due to the asymmetric helicase structure with a seam between subunits A and F. In each binding pose, the ssDNA emerging from the helicase consistently passes between the ZBD and RPD of primase.

Recent Publications

• Feng X^#, Spiering MM^#, Santos RLA, Benkovic SJ, Li H. Structural basis of the T4 bacteriophage primosome assembly and primer synthesis. BioRxiv 539249 [Preprint]. May 03, 2023. Available from: https://doi.org/10.1101/2023.05.03.539249. # These authors contributed equally to this work.

Is leading- and lagging-strand DNA replication a coordinated or uncoordinated process?

A long-standing important question is how leading- and lagging-strand DNA replication of a genome such as T4 is completed simultaneously. Observations reveal that the leading- and lagging-strand polymerases move at the same rate in both the E. coli and T4 systems. Consequently, one would anticipate that synthesis of the lagging strand would fall progressively behind that of the leading strand as replication continues due to the finite timescale of lagging-strand polymerase recycling. To explain this conundrum, hypotheses ranging from highly coordinated leading- and lagging-strand replication to completely independent and stochastic leading- and lagging-strand replication have been debated.

Using stopped-flow fluorescence kinetics and primer extension assays, we have shown that unused primer•primase complexes, which have a residence time on DNA similar to the time scale of Okazaki fragment synthesis, have the ability to block a progressing holoenzyme, and stimulate dissociation of the holoenzyme from DNA, triggering polymerase recycling (Figure 2). This mechanism for signaling has distinct advantages over those previously proposed because this signal does not require transmission to the lagging-strand polymerase through protein or DNA interactions. The mechanism for rapid dissociation of the holoenzyme is always collision, and no unique characteristics need to be assigned to either identical polymerase in the replisome. Using this signaling mechanism and the extensive experimental data for the T4 replisome, we carried out simulations of repeated cycles of Okazaki fragment initiation. The results indicate that initiation times vary during Okazaki fragment synthesis, during which Okazaki fragments can lengthen, where as unused primer•primase complexes provide a signaling mechanism by which Okazaki fragments can shorten. Collectively, this leads to a semi-random distribution of Okazaki fragment lengths that are consistent with available experimental data.

Figure 2. Proposed model for pppRNA–primase complex as the signal for lagging-strand initiation.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. A 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.

To follow up this study, we have devised an experiment to label and simultaneously distinguish between Okazaki fragments and gaps with high resolution (≤ ten nucleotides) and to then analyze the sequential order and lengths of the Okazaki fragments and gaps of the lagging strand synthesized by individual replisomes in a high-throughput manner to understand the coordination of leading- and lagging-strand DNA synthesis in the T4 replisome.

Recent Publications

• Spiering MM, Hanoian P, Gannavaram S & Benkovic SJ. (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. USA 114(22):5635-5640.

Reviews

• Benkovic SJ & Spiering MM. (2017) Understanding DNA replication by the bacteriophage T4 replisome. J. Biol. Chem. 292(45):18434-18442.
• Noble E, Spiering MM & Benkovic SJ. (2015) Coordinated DNA Replication by the Bacteriophage T4 Replisome. Viruses 7(6):3186-200.