Simultaneous single-molecule measurements of phage T7 replisome composition and function reveal the mechanism of polymerase exchange

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

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized λ DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2)

Structural and functional probing of PorZ, an essential bacterial surface component of the type-IX secretion system of human oral-microbiomic Porphyromonas gingivalis.

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

Artificial intelligence and the analysis of cryo-EM data provide structural insight into the molecular mechanisms underlying LN-lamininopathies

Abstract Laminins (Lm) are major components of basement membranes (BM), which polymerize to form a planar lattice on cell surface. Genetic alternations of Lm affect their oligomerization patterns and lead to failures in BM assembly manifesting in a group of human disorders collectively defined as Lm N-terminal domain lamininopathies (LN-lamininopathies). We have employed a recently determined cryo-EM structure of the Lm polymer node, the basic repeating unit of the Lm lattice, along with structure prediction and modeling to systematically analyze structures of twenty-three pathogenic Lm polymer nodes implicated in human disease. Our analysis provides the detailed mechanistic explanation how Lm mutations lead to failures in Lm polymerization underlining LN-lamininopathies. We propose the new categorization scheme of LN-lamininopathies based on the insight gained from the structural analysis. Our results can help to facilitate rational drug design aiming in the treatment of Lm deficiencies

Exploring the Structural Variability of Dynamic Biological Complexes by Single-Particle Cryo-Electron Microscopy

Biological macromolecules and assemblies precisely rearrange their atomic 3D structures to execute cellular functions. Understanding the mechanisms by which these molecular machines operate requires insight into the ensemble of structural states they occupy during the functional cycle. Single-particle cryo-electron microscopy (cryo-EM) has become the preferred method to provide near-atomic resolution, structural information about dynamic biological macromolecules elusive to other structure determination methods. Recent advances in cryo-EM methodology have allowed structural biologists not only to probe the structural intermediates of biochemical reactions, but also to resolve different compositional and conformational states present within the same dataset. This article reviews newly developed sample preparation and single-particle analysis (SPA) techniques for high-resolution structure determination of intrinsically dynamic and heterogeneous samples, shedding light upon the intricate mechanisms employed by molecular machines and helping to guide drug discovery efforts

Novel Artificial Intelligence-Based Approaches for Ab Initio Structure Determination and Atomic Model Building for Cryo-Electron Microscopy

Single particle cryo-electron microscopy (cryo-EM) has emerged as the prevailing method for near-atomic structure determination, shedding light on the important molecular mechanisms of biological macromolecules. However, the inherent dynamics and structural variability of biological complexes coupled with the large number of experimental images generated by a cryo-EM experiment make data processing nontrivial. In particular, ab initio reconstruction and atomic model building remain major bottlenecks that demand substantial computational resources and manual intervention. Approaches utilizing recent innovations in artificial intelligence (AI) technology, particularly deep learning, have the potential to overcome the limitations that cannot be adequately addressed by traditional image processing approaches. Here, we review newly proposed AI-based methods for ab initio volume generation, heterogeneous 3D reconstruction, and atomic model building. We highlight the advancements made by the implementation of AI methods, as well as discuss remaining limitations and areas for future development

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

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

Coupling dTTP hydrolysis with DNA unwinding by the DNA helicase of bacteriophage T7

The DNA helicase encoded by gene 4 of bacteriophage T7 assembles on single-stranded DNA as a hexamer of six identical subunits with the DNA passing through the center of the toroid. The helicase couples the hydrolysis of dTTP to unidirectional translocation on single-stranded DNA and the unwinding of duplex DNA. Phe523, positioned in a β-hairpin loop at the subunit interface, plays a key role in coupling the hydrolysis of dTTP to DNA unwinding. Replacement of Phe523 with alanine or valine abolishes the ability of the helicase to unwind DNA or allow T7 polymerase to mediate strand-displacement synthesis on duplex DNA. In vivo complementation studies reveal a requirement for a hydrophobic residue with long side chains at this position. In a crystal structure of T7 helicase, when a nucleotide is bound at a subunit interface, Phe523 is buried within the interface. However, in the unbound state, it is more exposed on the outer surface of the helicase. This structural difference suggests that the β-hairpin bearing the Phe523 may undergo a conformational change during nucleotide hydrolysis. We postulate that upon hydrolysis of dTTP, Phe523 moves from within the subunit interface to a more exposed position where it contacts the displaced complementary strand and facilitates unwinding

Cryo-EM reveals the molecular basis oflaminin polymerization and LN-lamininopathies

In this work the authors report the cryo-EM structure of the laminin polymer node and reveal the molecular basis of calcium-dependent formation of laminin lattice. The work provides insights into laminin polymerization defects manifesting in human disease