Thioredoxin suppresses microscopic hopping of T7 DNA polymerase on duplex DNA

Abstract

The DNA polymerases involved in DNA replication achieve high processivity of nucleotide incorporation by forming a complex with processivity factors. A model system for replicative DNA polymerases, the bacteriophage T7 DNA polymerase (gp5), encoded by gene 5, forms a tight, 1∶1 complex with Escherichia coli thioredoxin. By a mechanism that is not fully understood, thioredoxin acts as a processivity factor and converts gp5 from a distributive polymerase into a highly processive one. We use a single-molecule imaging approach to visualize the interaction of fluorescently labeled T7 DNA polymerase with double-stranded DNA. We have observed T7 gp5, both with and without thioredoxin, binding nonspecifically to double-stranded DNA and diffusing along the duplex. The gp5/thioredoxin complex remains tightly bound to the DNA while diffusing, whereas gp5 without thioredoxin undergoes frequent dissociation from and rebinding to the DNA. These observations suggest that thioredoxin increases the processivity of T7 DNA polymerase by suppressing microscopic hopping on and off the DNA and keeping the complex tightly bound to the duplex.T he bacteriophage T7 replisome is an elegantly simple, wellstudied model system of DNA replication (1). Replicative DNA synthesis is supported by T7 DNA polymerase (gp5), in a tight, 1∶1 complex with Escherichia coli thioredoxin, which will henceforth be referred to as gp5/trx. By itself, the 80-kDa gp5 synthesizes DNA in a distributive fashion and is capable of adding only a few nucleotides to the 3 0 end of a primer before dissociating from the primer template (2). Association of gp5 with the 12-kDa E. coli host protein thioredoxin results in a stable 1∶1 complex (K D ¼ 5 nM) and drastically increases the processivity of DNA synthesis. This interaction is critical for replication of T7 DNA-the phage cannot reproduce in thioredoxin null strains of E. coli (3, 4). The affinity of gp5/trx for primer-template DNA is increased at least 20-fold compared to that of gp5 alone (5), resulting in a complex that is capable of polymerizing hundreds of nucleotides before dissociation (2).Thioredoxin binds to gp5 via a 71-amino acid loop that resides between two alpha helices in the thumb domain and is called the thioredoxin-binding domain (TBD) (6). This loop is absent from other members of the Pol I family of DNA polymerases, but when inserted into E. coli DNA polymerase I at the homologous site, the resulting chimera displays a thioredoxin-dependent increase in processivity (7). The TBD also functions as a scaffold for the assembly of the replisome. The binding of thioredoxin structures the domain and creates two small solvent exposed loops containing binding sites for the T7 single-stranded DNA-binding protein and the T7 helicase (8, 9). Thioredoxin itself does not bind to DNA, and only one residue on thioredoxin has been implicated in binding to any of the other replisome components other than gp5 (10).Crystal structures of gp5/trx bound to primer-template DNA reveal that the TBD is extended over the dup...

Single-molecule studies of polymerase dynamics and stoichiometry at the bacteriophage T7 replication machinery

“…Polymerase labeling reactions were performed according to the procedure described by Etson et al (39) with an excess of either Alexa Fluor 488 carboxylic acid succinimidyl ester (Invitrogen) or Cy5 NHS ester (Lumiprobe) for an hour at room temperature. The conjugation reactions were biased to the N-terminal groups by adjusting the pH to 7.6.…”

It Seems Like Only Yesterday

“…These studies and the proper credits are well recorded in not-too-distant reviews (148)(149)(150)(151). The outstanding individuals who participated in these studies and are mentioned only briefly elsewhere include Barak Akabayov (143,(152)(153)(154)(155)(156)(157)(158), Nathalie Andraos (159) (149,180,181,(186)(187)(188)(189)(190)(191)(192)(193)(194)(195)(196)(197), Zhengguo He (198,199), Alfredo Hernandez (200), Anna Hine (201,202), Edel Hyland (203), Don Johnson (157,168,187,190,204,205), Young-Tae Kim (206)(207)(208)(209), Robin Kremsdorf (210), Arek Kulczyk (143,153,155,183,…”

“…J.B. Lee determined the rate of movement of the polymerase and helicase and showed that primer synthesis temporarily halts leading-strand synthesis (191). C. Etson used these techniques to show that thioredoxin, the processivity factor, suppresses microscopic hopping by the polymerase (186). Sam Hamdan, a research fellow in my lab, joined Antoine's group and showed that leading-and lagging-strand synthesis was coordinated with the formation and resolution of replication loops (188).…”

Bacterial replication, transcription and translation: mechanistic insights from single-molecule biochemical studies

“…The T7 system is even simpler, comprising only a polymerase (gp5), a combined helicase-primase (gp4) and an SSB (gp2.5) 30 . The Escherichia coli host protein thioredoxin is bound by the T7 polymerase and used as a processivity factor 78 . Single-molecule experiments have revealed key aspects of the T4 and T7 replication mechanisms that are likely to be conserved in bacteria.…”