A complete understanding of the molecular mechanisms underlying the functioning of large, multiprotein complexes requires experimental tools capable of simultaneously visualizing molecular architecture and enzymatic activity in real time. We developed a novel single-molecule assay that combines the flow-stretching of individual DNA molecules to measure the activity of the DNA-replication machinery with the visualization of fluorescently labeled DNA polymerases at the replication fork. By correlating polymerase stoichiometry with DNA synthesis of T7 bacteriophage replisomes, we are able to quantitatively describe the mechanism of polymerase exchange. We find that even at relatively modest polymerase concentration (∼2 nM), soluble polymerases are recruited to an actively synthesizing replisome, dramatically increasing local polymerase concentration. These excess polymerases remain passively associated with the replisome through electrostatic interactions with the T7 helicase for ∼50 s until a stochastic and transient dissociation of the synthesizing polymerase from the primer-template allows for a polymerase exchange event to occur.bacteriophage T7 | DNA replication | flow stretching | fluorescence T he replisome, a multiprotein complex that carries out DNA replication, is thought of as a stable molecular machine that duplicates genomic DNA in a rapid and continuous fashion (1). Even though high processivities and rates are observed in vitro (2), replisomes in a cellular environment face numerous obstacles including transcription complexes and DNA lesions that may stall the replicative polymerase (3). Recent work has challenged the notion that a single, unchanging replisome is able to replicate a complete genome in vivo (4, 5). The discovery of translesion DNA polymerases, specialized enzymes capable of synthesizing across damaged DNA bases, and novel mechanisms for replication restart suggest that the replisome might be highly dynamic, with components exchanging and entire replisomes collapsing and reassembling (6).Biochemical experiments have provided, at least at first glance, contradictory information regarding the stability of a key component of the replisome, DNA polymerases. Textbook models have portrayed the leading-strand polymerase as remaining tightly associated with the replisome, synthesizing DNA in a continuous fashion. On the lagging strand, the regular and rapid completion of Okazaki fragments requires either the recycling of the laggingstrand polymerase to the new RNA primer or the recruitment of a new polymerase from solution. Experiments have revealed that the replisomes of Escherichia coli, as well as the T4 and T7 bacteriophages remain highly processive even when challenged by dilution (7-10). At the same time, however, the T4 and T7 leading-and lagging-strand DNA polymerases exchange rapidly when challenged with excess polymerase in solution (11,12). Similar results have been obtained in E. coli, where translesion DNA polymerases are able to replace DNA polymerase III at both lesions and on u...

Porphyromonas gingivalis is a member of the human oral microbiome abundant in dysbiosis and implicated in the pathogenesis of periodontal (gum) disease. It employs a newly described type-IX secretion system (T9SS) for secretion of virulence factors. Cargo proteins destined for secretion through T9SS carry a recognition signal in the conserved C-terminal domain (CTD), which is removed by sortase PorU during translocation. Here, we identified a novel component of T9SS, PorZ, which is essential for surface exposure of PorU and posttranslational modification of T9SS cargo proteins. These include maturation of enzyme precursors, CTD removal and attachment of anionic lipopolysaccharide for anchorage in the outer membrane. The crystal structure of PorZ revealed two β-propeller domains and a C-terminal β-sandwich domain, which conforms to the canonical CTD architecture. We further documented that PorZ is itself transported to the cell surface via T9SS as a full-length protein with its CTD intact, independently of the presence or activity of PorU. Taken together, our results shed light on the architecture and possible function of a novel component of the T9SS. Knowledge of how T9SS operates will contribute to our understanding of protein secretion as part of host-microbiome interactions by dysbiotic members of the human oral cavity.

Replication of DNA plays a central role in transmitting hereditary information from cell to cell. To achieve reliable DNA replication, multiple proteins form a stable complex, known as the replisome, enabling them to act together in a highly coordinated fashion. Over the past decade, the roles of the various proteins within the replisome have been determined. Although many of their interactions have been characterized, it remains poorly understood how replication proteins enter and leave the replisome. In this study, we visualize fluorescently labeled bacteriophage T7 DNA polymerases within the replisome while we simultaneously observe the kinetics of the replication process. This combination of observables allows us to monitor both the activity and dynamics of individual polymerases during coordinated leading-and lagging-strand synthesis. Our data suggest that lagging-strand polymerases are exchanged at a frequency similar to that of Okazaki fragment synthesis and that two or more polymerases are present in the replisome during DNA replication. Our studies imply a highly dynamic picture of the replisome with lagging-strand DNA polymerases residing at the fork for the synthesis of only a few Okazaki fragments. Further, new lagging-strand polymerases are readily recruited from a pool of polymerases that are proximally bound to the replisome and continuously replenished from solution.polymerase exchange | single molecule | fluorescence microscopy T he organization of replisomes is highly conserved among various organisms (1), underlining the evolutionary importance of the replication machinery architecture. The bacteriophage T7 replication system offers an attractive model system to study the interplay between replication proteins because its replication machinery is relatively simple; a functional replisome can be reconstituted by just four purified proteins. Three of these proteins are encoded by the phage itself: helicase-primase (gp4), DNA polymerase (gp5), and single-stranded DNA (ssDNA) binding protein (gp2.5). A processivity factor for the gp5 polymerase, thioredoxin (trx), is provided by the host Escherichia coli.

. (2016). Simultaneous real-time imaging of leading and lagging strand synthesis reveals the coordination dynamics of single replisomes. Molecular Cell, 64 (6), 1035-1047.Simultaneous real-time imaging of leading and lagging strand synthesis reveals the coordination dynamics of single replisomes AbstractThe molecular machinery responsible for DNA replication, the replisome, must efficiently coordinate DNA unwinding with priming and synthesis to complete duplication of both strands. Due to the anti-parallel nature of DNA, the leading strand is copied continuously, while the lagging strand is produced by repeated cycles of priming, DNA looping, and Okazaki-fragment synthesis. Here, we report a multidimensional single-molecule approach to visualize this coordination in the bacteriophage T7 replisome by simultaneously monitoring the kinetics of loop growth and leading-strand synthesis. We show that loops in the lagging strand predominantly occur during priming and only infrequently support subsequent Okazaki-fragment synthesis. Fluorescence imaging reveals polymerases remaining bound to the lagging strand behind the replication fork, consistent with Okazaki-fragment synthesis behind and independent of the replication complex. Individual replisomes display both looping and pausing during priming, reconciling divergent models for the regulation of primer synthesis and revealing an underlying plasticity in replisome operation. SummaryThe molecular machinery responsible for DNA replication, the replisome, must efficiently coordinate DNA unwinding with priming and synthesis to complete duplication of both strands. Due to the anti-parallel nature of DNA, the leading strand is copied continuously, while the lagging strand is produced by repeated cycles of priming, DNA looping, andOkazaki-fragment synthesis. Here, we report a multidimensional single-molecule approach to visualize this coordination in the bacteriophage T7 replisome by simultaneously monitoring the kinetics of loop growth and leading-strand synthesis. We show that loops in the lagging strand predominantly occur during priming and only infrequently support subsequent Okazaki-fragment synthesis. Fluorescence imaging reveals polymerases remaining bound to the lagging strand behind the replication fork, consistent with Okazakifragment synthesis behind and independent of the replication complex. Individual replisomes display both looping and pausing during priming, reconciling divergent models for the regulation of primer synthesis and revealing an underlying plasticity in replisome operation.3

Gene 5 of bacteriophage T7 encodes a DNA polymerase (gp5) responsible for the replication of the phage DNA. Gp5 polymerizes nucleotides with low processivity, dissociating after the incorporation of 1 to 50 nucleotides. Thioredoxin (trx) of Escherichia coli binds tightly (Kd ¼ 5 nM) to a unique segment in the thumb subdomain of gp5 and increases processivity. We have probed the molecular basis for the increase in processivity. A single-molecule experiment reveals differences in rates of enzymatic activity and processivity between gp5 and gp5/trx. Small angle X-ray scattering studies combined with nuclease footprinting reveal two conformations of gp5, one in the free state and one upon binding to trx. Comparative analysis of the DNA binding clefts of DNA polymerases and DNA binding proteins show that the binding surface contains more hydrophobic residues than other DNA binding proteins. The balanced composition between hydrophobic and charged residues of the binding site allows for efficient sliding of gp5/trx on the DNA. We propose a model for trx-induced conformational changes in gp5 that enhance the processivity by increasing the interaction of gp5 with DNA. Bacteriophage T7 has evolved an efficient system for replicating its DNA (1). Four proteins account for most of the reactions that occur at the replication fork. Three of these proteins are encoded by the phage and one by the host. Therefore, phage T7 is able to bypass the more complicated Escherichia coli machinery. The four proteins are gene 5 DNA polymerase (gp5), E. coli thioredoxin (trx) processivity factor, gene 4 helicaseprimase, and gene 2.5 ssDNA binding protein.The crystal structure of gp5 in complex with trx, a primer template, and a nucleoside 5′-triphosphate in a polymerization mode is available (Fig. 1). The processivity factor, trx, binds tightly (5 nM) (2) in a one-to-one stoichiometry to a unique polypeptide loop (trx-binding domain) located in the thumb subdomain of gp5 (3) (Fig. 1). The binding of trx converts gp5 to a processive polymerase that polymerizes hundreds of nucleotides before dissociation from the DNA (4, 5). High processivity is an important property of replicative DNA polymerases that display high fidelity and a high rate of nucleotide polymerization (6). Two parameters that affect the processivity of DNA polymerases are often neglected: salt concentration and the sequence of the DNA template (6, 7).How does trx increase the processivity of gp5? The crystal structure of gp5/trx bound to a primer template shows the trxbinding domain with the associated trx extended over the duplex region of the primer template (7). The trx-binding domain (TBD) and trx do not completely encircle the DNA as the other half of the DNA is resting in the DNA binding crevice of gp5. Nevertheless, the result is a clamp-like structure. The binding of trx most likely also orients the trx-binding domain so that additional basic residues contact the phosphodiester backbone of the DNA leading to a higher affinity for the DNA through increased electrosta...

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Background:Interactions of DNA polymerase and DNA helicase are crucial in DNA synthesis. Results: Two distinct interactions are involved in formation of the DNA polymerase/DNA helicase complex. Conclusion:The multiple interactions between DNA polymerase and DNA helicase account for the high processivity of leading strand synthesis. Significance: Understanding of the replication process in bacteriophage T7 facilitates studies in more complex systems.

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