Structures and operating principles of the replisome

Abstract

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 and discontinuously (via Okazaki fragments) on the lagging strand, where each DNA synthesis initiates from an RNA primer provided by primase. After six decades, how DNA polymerases, helicase, primase, and their accessory factors form a replisome and perform concerted leading and lagging strand synthesis at a replication fork had never been visualized in atomic detail. RATIONALE Bacteriophage T7 presents the simplest known DNA replication system, consisting of only three proteins. Helicase and primase reside in one polypeptide chain that forms a hexamer in the presence of DNA and ATP or dTTP. T7 DNA polymerase, aided by E. coli thioredoxin as its processivity factor, carries out both leading and lagging strand DNA synthesis. Based on published biochemical data, we designed a minimal DNA fork to trap these essential proteins in replication competent states. RESULTS We determined cryogenic-electron microscopy (cryo-EM) structures of the T7 replisome and showed how its essential enzymatic functions are coordinated in three dimensions. The hexameric helicase adopts a spiral “lock washer” form that encircles the coil-like lagging DNA strand, with two nucleotides (nt) bound to each protein subunit and adjacent helicase subunits linked by domain swapping. ATP hydrolysis propels each helicase domain to translocate sequentially and coaxially along DNA in a hand-over-hand fashion, advancing 2 nt per step in the 5′ to 3′ direction (Fig. A). Instead of all enzymes 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 unspool the downstream DNA tangentially (Fig. B). 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, synthesizes 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 (Fig. B). 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, bind two amino-acid residues with each su...

Replication fork pausing at protein barriers on chromosomes

“…Replicative helicases, including gp4 in bacteriophage T7 , DnaB in Escherichia coli , MCM homo‐hexamer–Cdc45/RecJ‐like protein–GINS complex in archaea , and Cdc45, Mcm2–7 hexamer and GINS complex (CMG complex) in eukaryotes , have a ring‐like shape and are considered to hold unwound ssDNA in the hole of the ring . The helicases bound stably to DNA associate with DNA polymerase, which also binds to unwound ssDNA for DNA synthesis and interacts with a clamp complex, the β clamp in E. coli , or PCNA in eukaryotes. In this way, the replication machineries establish the stable association with template DNA.…”

DNA unwinding mechanism of a eukaryotic replicative CMG helicase

“…In a recently reported cryo-EM structure of the T7 replisome complexed with a forked DNA, only ssDNA enters the helicase ring at the C-tier, with no interaction between the helicase and the parental duplex (Gao et al, 2019). Interestingly, the dsDNA and ssDNA orient perpendicularly to one another, and this geometry is hypothesized to greatly accelerate the rate of T7 helicase activity (Gao et al, 2019).…”

“…While bacterial replicative helicases travel 5'-3' with the C-tier motors leading the way, eukaryotic helicases track 3'-5' on DNA with the N-face in front, pushed by motors in the C-tier (Enemark and Joshua-Tor, 2008;Li and O'Donnell, 2018;O'Donnell and Li, 2018). The way in which helicases engage ssDNA in the motor domains has been documented for several different hexameric replicative helicases and involve binding to loops in the ATPase domains (Abid Ali et al, 2017;Enemark and Joshua-Tor, 2006;Gao et al, 2019;Itsathitphaisarn et al, 2012;Li and O'Donnell, 2018;Meagher et al, 2019;O'Donnell and Li, 2018;Skordalakes and Berger, 2006;Thomsen and Berger, 2009). In eukaryotes the main loop is referred to as PS1 (e.g.…”

CryoEM structures of human CMG - ATPγS - DNA and CMG - AND-1 complexes

“…Overall, the observations relative to ATP status and DNA binding in the MCM C-tier ring are consistent with a sequential rotary mechanism of ATP hydrolysis as the basis for translocation of the human CMG. The structural integrity of the C-tier during translocation is provided by a domain-swapped helix (Supplementary figure 4) that provides a flexible tether between contiguous MCM subunits, in a similar fashion as recently described for the replicative gp4 DNA helicase (Gao et al, 2019).…”

“…The mechanism of translocation by which the CMG couples ATP hydrolysis to processive DNA unwinding is the current focus of intense research efforts. Based on structural analysis of bacteriophage, viral and bacterial systems (Enemark & Joshua-Tor, 2006;Gao et al, 2019;Itsathitphaisarn, Wing, Eliason, Wang, & Steitz, 2012;Singleton, Sawaya, Ellenberger, & Wigley, 2000) a consensus has emerged for a sequential rotary mechanism of DNA unwinding by replicative DNA helicases. In this mechanism, ATP is sequentially hydrolysed by successive ring subunits so that each ring position cycles through ATP, ADP and apo states.…”