DNA Replication

DNA replication: The double helix is un'zipped' and unwound, then each separated strand (turquoise) acts as a template for replicating a new partner strand (green). Nucleotides (bases) are matched to synthesize the new partner strands into two new double helices. ¹

Eukaryotes are organisms whose cells have a nucleus enclosed within membranes, unlike prokaryotes (Bacteria and Archaea), which have no membrane-bound organelles. ²

INTRODUCTION
DNA replication has been studied since the 1950s. It is well established that double-helical DNA needs to be separated for replication by a helicase. … Six decades after the discovery of the DNA double helix, visualization in atomic detail of how a functional replisome is formed and performs concerted leading- and lagging-strand synthesis at a replication fork has not been reported. ³

We determined cryo–electron microscopy (cryo-EM) structures of the T7 replisome and show how its essential enzymatic functions are coordinated in three dimensions. ⁴

CONCLUSION
We note the similarity between hexameric DNA helicases and AAA+ protein chaperones and unfoldases, which form spiral-shaped hexamers around protein substrates and move along proteins by a hand-over-hand subunit translocation mechanism. The operating principles of the bacteriophage replisome observed here rationalize many well-known features of bacterial and eukaryotic replication. In each replisome, a helicase is the central organizer and tangentially unspools the downstream DNA, while leading- and lagging-strand polymerases synthesize DNA separately on the front and back sides of the helicase. ⁵

On page 835 of this issue, Gao et al.,  report the cryo–electron microscopy (cryo-EM) structure of the T7 bacteriophage replisome at a high atomic detail. The study not only advances our understanding of the helicase mechanism but also reveals an unexpected arrangement of the two DNA Pols in the replisome. Specifically, the two Pols sandwich the DNA helicase in an asymmetric manner; one DNA Pol is on top of the helicase, and one DNA Pol is below (see the figure). This architecture is unlike textbook illustrations of both DNA Pols trailing behind the helicase. ⁶

Replication of the DNA genome is performed by a replisome complex composed of numerous proteins. Cells have duplex DNA genomes, and their replisomes duplicate both strands simultaneously. A functional replisome requires, at a minimum, a helicase to unwind the DNA duplex, two DNA polymerases (Pols) to replicate the two DNA strands, and a primase to form RNA primers that DNA Pols extend. The replisome functions at a Y junction, or replication fork, and is a complex task because DNA Pols can only extend DNA in a 3'-to-5' direction. Thus, as the helicase unwinds the antiparallel DNA strands, the DNA Pol on one strand (the leading strand) can go in the same direction as the helicase and replicate DNA continuously, but the DNA Pol on the antiparallel strand (the lagging strand) is generated in the opposite direction. This requires repeated priming and extension of the lagging strand discontinuously as a series of Okazaki fragments. This “semidiscontinuous replication” is shared by all cells. On page 835 of this issue, Gao et al. (1) report the cryo–electron microscopy (cryo-EM) structure of the T7 bacteriophage replisome at a high atomic detail. The study not only advances our understanding of the helicase mechanism but also reveals an unexpected arrangement of the two DNA Pols in the replisome. Specifically, the two Pols sandwich the DNA helicase in an asymmetric manner; one DNA Pol is on top of the helicase, and one DNA Pol is below (see the figure). This architecture is unlike textbook illustrations of both DNA Pols trailing behind the helicase. ⁷

Multiprotein complexes carry out each step of the “central dogma” of genetic information flow: replication, transcription, and translation. Interestingly, proteins of transcription and translation are homologous among Bacteria, Archaea, and Eukarya and thus evolved from a common ancestor. By contrast, the DNA Pol, helicase, and primase of bacterial replisomes share no homology to their eukaryotic counterparts, implying that these replisome enzymes evolved independently, after the evolutionary split of bacteria and eukaryotes. The primordial cell possibly used a simpler process of DNA replication, or used an RNA genome.

Surprisingly, the asymmetric arrangement of two DNA Pols that sandwich the helicase was also demonstrated by EM for the eukaryotic replisome of the yeast Saccharomyces cerevisiae, albeit at lower resolution than the T7 study by Gao et al. Thus, although “worlds apart” in terms of their independent evolution, the core elements of bacterial and eukaryotic replisomes both contain a helicase between two DNA Pols, although the eukaryotic replisome requires a trimeric scaffolding factor (Ctf4) to help tether the top DNA Pol to the helicase.

Another unexpected feature of the bacterial (T7) and eukaryotic replisomes is that the top DNA Pol functions on the opposite strand: The DNA Pol at the top of T7 helicase replicates the leading strand, whereas the DNA Pol at the top of eukaryotic CMG helicase replicates the lagging strand (see the figure). This is because bacterial helicases encircle the lagging strand, whereas eukaryotic CMG helicase encircles the leading strand. It remains a mystery why this “mirror” arrangement evolved, but both arrangements share a pragmatic logic for replisome function. All replicative helicases split the duplex at their leading edge, with one strand going through the middle of the helicase ring and the other strand deflected off the top of the ring. Hence, a DNA Pol at the top of the helicase can immediately duplicate the separated strand. ⁶