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.
Replisomes require core enzymes: helicase, primase, and DNA polymerases (Pols). Replisomes of both bacteria and eukaryotes require many more proteins not shown here. Textbook illustrations have placed both leading- and lagging-strand DNA Pols behind the helicase. Insights from Gao et al.reveal the asymmetric juxtaposition of these core enzymes in T7 phage, similar to replisiomes in the eukaryote Saccharomyces cerevisiae.
GRAPHIC: N. DESAI/SCIENCE
T7 phage is a smart choice for cryo-EM studies because it is the most streamlined replisome known (2). The helicase in T7 phage, gene protein 4 (gp4), forms a hexameric ring, a common feature of all replicative helicases (3, 4). However, unlike other helicases, T7 gp4 also contains a primase located behind the helicase during replication fork progression (2). The T7 DNA Pol, gp5, functions in a 1:1 complex with the 12-kDa bacterial host thioredoxin protein, which increases polymerase processivity (2). Hence, three different proteins comprise the core T7 replisome (gp4, gp5, and thioredoxin). By contrast, the replisome of Escherichia coli—the bacterial host for T7—contains a dozen different proteins, including a sliding clamp and clamp loader complex for replisome processivity (5).
Despite the different complexity, the proteins of the T7 replisome are homologous to proteins of its E. coli host and thus are representative of the bacterial core replisome. Gao et al. report the 3.2-Å resolution structure of the T7 replisome in which the gp4 helicase-primase encircles the lagging strand to unwind the parental duplex while also priming the lagging strand. The leading- and lagging-strand DNA Pols connect to and sandwich the helicase between them, an asymmetric arrangement that minimizes single-stranded DNA (ssDNA), which is more susceptible to nucleases and damage than double-stranded DNA (dsDNA).
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 (6, 7). 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 (8), 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 (9–13). 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 (4). Hence, a DNA Pol at the top of the helicase can immediately duplicate the separated strand.
In bacteria, the primase domain of gp4 is below the helicase for Okazaki fragment extension. In eukaryotes, priming is performed above the CMG helicase by the Pol α–primase complex, which is secured to the helicase by the Ctf4 scaffold (8, 12–14). Pol α–primase generates an RNA-DNA primer of ∼25 nucleotides for the lagging strand Pol δ (13, 14). In eukaryotes, the leading and lagging DNA Pols are encoded by different genes, and the leading strand DNA Pol ε (14) is located at the bottom of the helicase, which encircles the leading strand and feeds the unwound strand to Pol ε for continuous leading-strand synthesis (8, 11).
There exist numerous questions for the future. Having a DNA Pol on top of the helicase in T7 suggests that it may be first to encounter DNA damage and DNA-bound proteins before the helicase. Therefore, the DNA Pol arrangement may help replisome progression by detecting lesions or displacing barriers. Single-molecule studies indicate a network of dynamic enzyme exchanges within the replisome of T7 and other replisomes, which may eject or supplement DNA Pols as needed (15). There is much excitement over these new discoveries, and there are many more questions to address in the future than have thus far been answered.
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