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.

SMC (structural maintenance of chromosome) proteins act ubiquitously in chromosome processing. In Escherichia coli, the SMC complex MukBEF plays roles in chromosome segregation and organization. We used single-molecule millisecond multicolor fluorescence microscopy of live bacteria to reveal that a dimer of dimeric fluorescent MukBEF molecules acts as the minimal functional unit. On average, 8 to 10 of these complexes accumulated as "spots" in one to three discrete chromosome-associated regions of the cell, where they formed higher-order structures. Functional MukBEF within spots exchanged with freely diffusing complexes at a rate of one complex about every 50 seconds in reactions requiring adenosine triphosphate (ATP) hydrolysis. Thus, by functioning in pairs, MukBEF complexes may undergo multiple cycles of ATP hydrolysis without being released from DNA, analogous to the behavior of well-characterized molecular motors.

SummaryA prevalent view of DNA replication has been that it is carried out in fixed “replication factories.” By tracking the progression of sister replication forks with respect to genetic loci in live Escherichia coli, we show that at initiation replisomes assemble at replication origins irrespective of where the origins are positioned within the cell. Sister replisomes separate and move to opposite cell halves shortly after initiation, migrating outwards as replication proceeds and both returning to midcell as replication termination approaches. DNA polymerase is maintained at stalled replication forks, and over short intervals of time replisomes are more dynamic than genetic loci. The data are inconsistent with models in which replisomes associated with sister forks act within a fixed replication factory. We conclude that independent replication forks follow the path of the compacted chromosomal DNA, with no structure other than DNA anchoring the replisome to any particular cellular region.

Cellular DNA damage is reversed by balanced repair pathways that avoid accumulation of toxic intermediates. Despite their importance, the organization of DNA repair pathways and the function of repair enzymes in vivo have remained unclear because of the inability to directly observe individual reactions in living cells. Here, we used photoactivation, localization, and tracking in live Escherichia coli to directly visualize single fluorescent labeled DNA polymerase I (Pol) and ligase (Lig) molecules searching for DNA gaps and nicks, performing transient reactions, and releasing their products. Our general approach provides enzymatic rates and copy numbers, substrate-search times, diffusion characteristics, and the spatial distribution of reaction sites, at the single-cell level, all in one measurement. Single repair events last 2.1 s (Pol) and 2.5 s (Lig), respectively. Pol and Lig activities increased fivefold over the basal level within minutes of DNA methylation damage; their rates were limited by upstream base excision repair pathway steps. Pol and Lig spent >80% of their time searching for free substrates, thereby minimizing both the number and lifetime of toxic repair intermediates. We integrated these single-molecule observations to generate a quantitative, systems-level description of a model repair pathway in vivo.single-molecule tracking | super-resolution microscopy | DNA damage response | protein-DNA interaction | cytosolic diffusion A ll cellular organisms rely on complex DNA repair mechanisms for faithful chromosome replication and maintenance of their genome integrity (1). The variety of DNA lesions requires modular repair pathways that carry out damage recognition, damage removal, repair synthesis, and ligation in sequential steps catalyzed by a series of enzymes. However, all repair pathway steps need to be precisely balanced to avoid accumulation of DNA intermediates that are typically more mutagenic and toxic than the original lesion (2). Rapid processing of gapped and nicked intermediates is particularly crucial (3) because they provoke lethal double-strand breaks upon encountering replication forks (4); a single such break can lead to chromosome loss and cell death.Despite extensive genetic, biochemical, and biophysical studies (1), the molecular organization of DNA repair in vivo remains unclear. Most of our mechanistic understanding relies on in vitro ensemble studies, which cannot replicate the cellular environment and stochastic nature of chemical reactions. By avoiding ensembleaveraging, single-molecule experiments have revolutionized the study of protein-DNA interactions in vitro, but extension of these powerful concepts to DNA repair measurements in living cells remains an open goal. Early in vivo work focused on the mean behavior of cell populations and could not examine functionally important heterogeneity, such as the variation in protein copy numbers between cells and over time (5, 6). Such variation can lead to different repair rates across genetically identical cells and may dera...

SummaryThe circular Escherichia coli chromosome is organized by bidirectional replication into two equal left and right arms (replichores). Each arm occupies a separate cell half, with the origin of replication (oriC) at mid-cell. E. coli MukBEF belongs to the ubiquitous family of SMC protein complexes that play key roles in chromosome organization and processing. In mukBEF mutants, viability is restricted to low temperature with production of anucleate cells, reflecting chromosome segregation defects. We show that in mukB mutant cells, the two chromosome arms do not separate into distinct cell halves, but extend from pole to pole with the oriC region located at the old pole. Mutations in topA, encoding topoisomerase I, do not suppress the aberrant positioning of chromosomal loci in mukB cells, despite suppressing the temperature-sensitivity and production of anucleate cells. Furthermore, we show that MukB and the oriC region generally colocalize throughout the cell cycle, even when oriC localization is aberrant. We propose that MukBEF initiates the normal bidirectional organization of the chromosome from the oriC region.

A body of evidence supports the idea that newly replicated Escherichia coli chromosomes segregate progressively as replication progresses, with spatial separation of sister genetic loci occurring ∼15 min after their replication. We show that the time of this cohesion can be modulated by topoisomerase IV (TopoIV) activity. Impairment of TopoIV prevents segregation of newly replicated sister loci and bulk chromosome segregation, whereas modest increases in TopoIV decrease the cohesion time substantially. Therefore, we propose that precatenanes, which form as replication progresses by interwinding of newly replicated sister chromosomes, are responsible for E. coli sister chromosome cohesion. The heritable maintenance of the genetic material over generations requires not only that it be accurately replicated but that newly replicated chromosomes are faithfully transmitted to daughter cells at cell division. Defects in chromosome segregation are associated with genetic disease and cancer. In eukaryotes, newly replicated sister chromosomes remain associated and aligned until the onset of mitosis by dedicated cohesion mechanisms, using SMC (structural maintenance of chromosomes) proteins. Formation and dissolution of cohesion is under strict spatial and temporal control (for review, see Nasmyth and Haering 2005). Similarly, in the archaeon, Sulfolobus sulfataricus, sister cohesion seems to extend from the replicative to the post-replicative phase of the cell cycle (Robinson et al. 2007). In contrast, a growing body of evidence supports the view that newly replicated loci in bacteria segregate progressively as replication proceeds, with a period of sister cohesion (the time between locus replication and separation of the two sister loci) that is substantially less than S phase (Viollier et al. 2004; Neilsen et al. 2006;Reyes-Lamothe et al. 2008). Nevertheless, some work has led to the conclusion that extensive sister cohesion may hold sisters together for a large fraction of S phase (Sunako et al. 2001;Bates and Kleckner 2005).Topological entanglement of newly replicated sisters has been implicated in facilitating sister chromosome cohesion in bacteria, archaea, and eukaryotes, with precatenanes (interwound sister DNA duplexes present in a replicating chromosome), catenanes (interwound replicated sister duplexes), or hemicatenanes (duplexes inter- The right-hand (RH) interwinding of the two strands of a DNA duplex (linkage) has to be completely removed in order that newly replicated chromosomes can be segregated to daughter cells. Complete segregation of newly replicated Escherichia coli chromosomes requires the removal of ∼4.2 × 10 5 links in one generation time or less. RH precatenanes will arise when RH links in duplex DNA ahead of a progressing replication fork diffuse backward to behind the fork, thus interlinking the two newly replicated sisters by a redistribution of total linkage ( Fig. 1A; Champoux and Been 1980;Espeli and Marians 2004). In the absence of topoisomerase action, replication progression leads to a...

Once described as mere “bags of enzymes,” bacterial cells are in fact highly organized, with many macromolecules exhibiting nonuniform localization patterns. Yet the physical and biochemical mechanisms that govern this spatial heterogeneity remain largely unknown. Here, we identify liquid–liquid phase separation (LLPS) as a mechanism for organizing clusters of RNA polymerase (RNAP) in Escherichia coli. Using fluorescence imaging, we show that RNAP quickly transitions from a dispersed to clustered localization pattern as cells enter log phase in nutrient-rich media. RNAP clusters are sensitive to hexanediol, a chemical that dissolves liquid-like compartments in eukaryotic cells. In addition, we find that the transcription antitermination factor NusA forms droplets in vitro and in vivo, suggesting that it may nucleate RNAP clusters. Finally, we use single-molecule tracking to characterize the dynamics of cluster components. Our results indicate that RNAP and NusA molecules move inside clusters, with mobilities faster than a DNA locus but slower than bulk diffusion through the nucleoid. We conclude that RNAP clusters are biomolecular condensates that assemble through LLPS. This work provides direct evidence for LLPS in bacteria and demonstrates that this process can serve as a mechanism for intracellular organization in prokaryotes and eukaryotes alike.

In dividing cells, chromosome duplication once per generation must be coordinated with faithful segregation of newly replicated chromosomes and with cell growth and division. Many of the mechanistic details of bacterial replication elongation are well established. However, an understanding of the complexities of how replication initiation is controlled and coordinated with other cellular processes is emerging only slowly. In contrast to eukaryotes, in which replication and segregation are separate in time, the segregation of most newly replicated bacterial genetic loci occurs sequentially soon after replication. We compare the strategies used by chromosomes and plasmids to ensure their accurate duplication and segregation and discuss how these processes are coordinated spatially and temporally with growth and cell division. We also describe what is known about the three conserved families of ATP-binding proteins that contribute to chromosome segregation and discuss their inter-relationships in a range of disparate bacteria.