Stoichiometry and Architecture of Active DNA Replication Machinery in Escherichia coli

Abstract

The multiprotein replisome complex that replicates DNA, has been extensively characterized in vitro, but its composition and architecture in vivo is unknown. Using millisecond single molecule fluorescence microscopy in living cells expressing YPet derivatives of replisome components, we have examined replisome stoichiometry and architecture. Active Escherichia coli replisomes contain three molecules of the replicative polymerase, rather than the historically accepted two. These are associated with three molecules of τ, a clamp loader component that trimerizes polymerase. Only two of the three sliding clamps are always associated with the core replisome. Single strand binding protein has a broader spatial distribution than the core components, with five to eleven tetramers per replisome. This in vivo technique could provide single molecule insight into other molecular machines.Replisomes are dynamic multiprotein machines that replicate DNA by copying the leading strand template continuously and the lagging strand template discontinuously. In E. colithe replisome couples activities of more than 11 proteins during genome replication (1, 2). The DnaB helicase, loaded onto the lagging strand template, separates the two templates that are subsequently copied by PolIII polymerase ([αεθ). PolIII processivity results from binding to a sliding clamp (β) encircling duplex DNA; sliding clamps are added and removed by a clamp loader ([τ/γ] 3 δδ′ψχ) whose τ component oligomerizes PolIII. Unwound DNA on the lagging template strand is bound by single strand binding protein (Ssb) tetramers that remove DNA secondary structure and protect against nucleases. Primase binds to helicase during cycles of priming and DNA synthesis on the lagging strand template.

Analytical tools for single-molecule fluorescence imaging in cellulo

“…35 There is common agreement on a canonical shape for E. coli bacteria, namely that of a cylindrical body capped by hemispheres, with a typical diameter width of ~1 m and endto-end length in the range ~2-5 m, depending primarily on stage in the cell cycle. 7,8,[16][17][18] Here, we define a local coordinate system from each bacterium's position, orientation, length and width, as estimated from the non-fluorescence brightfield imaging data. Through observations using light microscopy we can characterize the size and shape of each detected E. coli cell individually, with its characteristic 'stubby' object shape 32 shown in Fig.…”

“…In essence, typical singlemolecule fluorescence imaging datasets have a poor equivalent signal-to-noise ratio (SNR) in reference to the typical pixel intensities registered on camera detectors, and are often relatively short in duration, for example consisting of 10 or less consecutive image frames. 7,8 However, it is also not uncommon to acquire significant quantities of such datasets during experimental runs, essential in constructing the underlying probability distribution of heterogeneous and stochastic molecular behaviour. The need for objective, robust, automated high-throughput and computationally efficient analysis tools is imperative to determine the underlying molecular properties that are markers of biochemical, physical chemical and chemical physics features of the internal cellular environment.…”

Transcription factors in eukaryotic cells can functionally regulate gene expression by acting in oligomeric assemblies formed from an intrinsically disordered protein phase transition enabled by molecular crowding

“…We used millisecond Slimfield single-molecule fluorescence imaging [44,45,51] on live S. cerevisiae cells (Figure 1(a ) ) using a green fluorescent protein (GFP) reporter for Mig1 integrated into the genome, including mCherry reporter on the RNA polymerase subunit protein Nrd1 to indicate the position of the cell nucleus. Slimfield was optimized for single-molecule detection sensitivity by using an in vitro imaging assay [52].…”

“…These single-molecule/cell and super-resolution microscopy tools have in particular been applied to integrated membrane proteins [29,30], such as interaction networks like oxidative phosphorylation [31–35], cell division processes [36–38] and protein translocation [39], along with bacterial cell motility [40–43]. The tools can also probe the aqueous environment of cells as opposed to just on their hydrophobic cell membrane surface, including processes of DNA replication/remodeling/repair [44–46], and systems more directly relevant to biomedicine such as bacterial infection [47–49]. …”

Bringing single-molecule spectroscopy to macromolecular protein complexes

“…Using a total internal reflection microscope, Ulbrich et al determined the subunit stoichiometry of membrane-bound proteins in Xenopus laevis oocytes by counting the number of GFP molecules [65], and Madl et al determined the stoichiometry of Orai1 channel proteins in mammalian cells by carrying out brightness analysis of GFP signals [66]. Using slimfield fluorescence microscopy, Reyes-Lamothe et al investigated the replisome stoichiometry and architecture in a bacteria [67]. Other examples of quantitative stoichiometry analysis are found in a review by Coffman and Wu [68].…”