Structures and operating principles of the replisome

Structures of the simplest replisome The DNA replisome performs concerted parental-strand separation and DNA synthesis on both strands. Gao et al. report the cryo–electron microscopy structures of the minimum set of bacteriophage T7 proteins that can carry out leading- and lagging-strand synthesis at the replication fork (see the Perspective by Li and O'Donnell). Three key enzymes involved in DNA replication—DNA polymerase, helicase, and primase—were visualized in complex with substrate DNA, demonstrating their highly dynamic organizations on both strands. Comparison of prokaryotic and eukaryotic replisomes reveals evolutionarily conserved operating principles and provides a structural basis for understanding coordination among DNA replication, recombination, and repair. Science, this issue p. eaav7003; see also p. 814 Cryo-EM structures of the bacteriophage T7 replisome carry out concerted leading- and lagging-strand DNA synthesis. 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. Each strand is then copied by a DNA polymerase, continuously on the leading strand and discontinuously (via Okazaki fragments) on the lagging strand, where each DNA synthesis initiates from an RNA primer provided by primase. In contrast to the transcription of DNA to RNA and the translation of messenger RNA to protein by ribosomes, which proceed on one track in one direction, in replication, the helicase and two DNA polymerases translocate along three different tracks, the parental DNA and the two separated antiparallel strands. The three moving components in a replisome are often depicted in textbooks as moving in the same direction parallel to the parental DNA. The absence of evolutionary conservation for DNA polymerases, helicases, primases, and accessory factors from bacteria to eukaryotes adds another layer of complication. Helicases involved in replication have different folds and translocate on the lagging strand in the 5′-to-3′ direction in bacteria and on the leading strand in the 3′-to-5′ direction in eukaryotes. Even the relatively simple bacteriophage T7 helicase can exist as a closed ring-shaped hexamer or heptamer or a coiled filament in the absence of DNA. Crystal structures of many polymerase-DNA complexes have been determined, but in previous attempts to obtain a structure of a polymerase and helicase complex, the DNA substrate is either partially or completely dissociated. 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. RATIONALE Bacteriophage T7 presents the simplest known DNA replisome, consisting of only three proteins. Helicase and primase reside in one polypeptide chain that forms a hexamer in the presence of DNA and adenosine triphosphate (ATP) or deoxythymidine triphosphate (dTTP). T7 DNA polymerase, aided by Escherichia coli thioredoxin as a processivity factor, carries out both leading- and lagging-strand DNA synthesis. On the basis of published biochemical data, we designed a minimal DNA fork to trap these essential proteins in replication competent states. RESULTS We determined cryo–electron microscopy (cryo-EM) structures of the T7 replisome and show how its essential enzymatic functions are coordinated in three dimensions. The hexameric helicase adopts a spiral “lock washer” form that encircles a coil-like DNA strand, with two nucleotides (nt) bound to each protein subunit. With adjacent helicase subunits linked by domain swapping, ATP hydrolysis propels each helicase domain to translocate sequentially along DNA in a hand-over-hand fashion, advancing 2 nt per step in the 5′-to-3′ direction (see the figure, top). Instead of all replisome components moving in the same direction parallel to the downstream parental DNA, a β hairpin from the leading-strand polymerase separates the two parental DNA strands into a T-shaped fork that enables the closely coupled helicase to move perpendicular to the downstream DNA and unspool it tangentially (see the figure, bottom). By protein-protein and DNA-mediated interactions, the leading-strand DNA polymerase and helicase cooperate to determine the rate of replication. For every ATP hydrolyzed and 2 nt advanced on DNA by the helicase, the DNA polymerase incorporates two deoxyribonucelotides. T7 primase, separated from the leading-strand polymerase by the helicase domain, generates the RNA primers needed to initiate lagging-strand DNA synthesis. Transfer of a short RNA primer from the primase to DNA polymerase is facilitated by a zinc-binding domain at the N terminus of the T7 primase-helicase protein. Two lagging-strand polymerases can be attached to the hexameric primases with one actively synthesizing DNA and the other waiting for a primer (see the figure, bottom). Such a relay system may allow the discontinuous lagging-strand synthesis to keep pace with the leading-strand synthesis. 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. Structure of T7 replisomes. (Top) A hand-over-hand mechanism for the translocation of hexameric helicase along a single-stranded DNA. Helicase subunits are indicated by the letters A to F. The numbers are a relative measure of the nucleotides that helicase translocates. (Bottom) In the T7 replisome, the leading-strand DNA polymerase (Pol) and helicase establish a T-shaped DNA replication fork. Primase supplies RNA primers for lagging-strand DNA polymerases to make Okazaki fragments in a relay mechanism. dNTP, deoxynucleotide triphosphate. Visualization in atomic detail of the replisome that performs concerted leading– and lagging–DNA strand synthesis at a replication fork has not been reported. Using bacteriophage T7 as a model system, we determined cryo–electron microscopy structures up to 3.2-angstroms resolution of helicase translocating along DNA and of helicase-polymerase-primase complexes engaging in synthesis of both DNA strands. Each domain of the spiral-shaped hexameric helicase translocates sequentially hand-over-hand along a single-stranded DNA coil, akin to the way AAA+ ATPases (adenosine triphosphatases) unfold peptides. Two lagging-strand polymerases are attached to the primase, ready for Okazaki fragment synthesis in tandem. A β hairpin from the leading-strand polymerase separates two parental DNA strands into a T-shaped fork, thus enabling the closely coupled helicase to advance perpendicular to the downstream DNA duplex. These structures reveal the molecular organization and operating principles of a replisome.