Rodrigo Reyes-Lamothe

Stoichiometry and Architecture of Active DNA Replication Machinery in Escherichia coli

Forking Replisomes Replisomes are multiprotein machines that replicate DNA. Significant insight into how they work comes from in vitro studies, but how replisomes are organized in living cells has remained unclear. Reyes-Lamothe et al. (p. 498) have watched the replisome in living Escherichia coli cells using single-molecule fluorescence spectroscopy with millisecond time resolution. Cells expressing fluorescent derivatives of 10 different replisome components revealed both the stoichiometry and spatial distribution of the components at active replication forks in Escherichia coli. A similar technique could be used to study other molecular machines as they function. Single-molecule fluorescence microscopy reveals the organization of the replisome in living bacterial cells. 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 fluorescent 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 5 to 11 tetramers per replisome. This in vivo technique could provide single-molecule insight into other molecular machines.

Independent Positioning and Action of Escherichia coli Replisomes in Live Cells

Summary A 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.

Chromosome Replication and Segregation in Bacteria

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.

In Vivo Architecture and Action of Bacterial Structural Maintenance of Chromosome Proteins

Making a Move Structural Maintenance of Chromosome (SMC) complexes act ubiquitously in chromosome processing in all domains of life, but their mode of action in living cells has remained an enigma. Badrinarayanan et al. (p. 528) used noninvasive millisecond single-molecule imaging to understand SMC complex molecular biochemistry in living bacterial cells with super-resolution spatial precision. Escherichia coli SMC complexes, which are important for chromosome segregation, formed dimers that bound to DNA in an adenosine triphosphate (ATP)–dependent manner and that could be released upon ATP-hydrolysis. By functioning in pairs, the complexes are likely to be able to undergo multiple cycles of ATP-hydrolysis without being released from DNA. SMC proteins form a dimer of adenosine triphosphate (ATP)–bound dimers, which translate ATP hydrolysis into mechanical DNA remodeling. 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.

MukB colocalizes with the oriC region and is required for organization of the two Escherichia coli chromosome arms into separate cell halves.

The 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.

Single-molecule DNA repair in live bacteria

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.

Escherichia coli and its chromosome

The Escherichia coli chromosome is a circular DNA molecule that is approximately 1000 times compacted in the living cell, where it occupies approximately 15% of the cellular volume. The genome is organized in a way that facilitates chromosome maintenance and processing. Despite huge efforts, until recently little has been known about how the chromosome is organized within cells, where replication takes place, and how DNA is segregated before cell division. New techniques for labeling genetic loci and molecular machines are allowing the simultaneous tracking of genetic loci and such machines in living cells over time. These studies reveal remarkable organization, yet a highly dynamic flux of genetic loci and macromolecules. It seems likely that the cellular positioning of chromosomal loci is the outcome of the formation of two chromosome arms (replichores) by replication, followed by sequential chromosome segregation, rather than from the presence of cellular positioning markers.

Modulation of Escherichia coli sister chromosome cohesion by topoisomerase IV.

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 approximately 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.

Replication and segregation of an Escherichia coli chromosome with two replication origins

Characterized bacteria, unlike eukaryotes and some archaea, initiate replication bidirectionally from a single replication origin contained within a circular or linear chromosome. We constructed Escherichia coli cells with two WT origins separated by 1 Mb in their 4.64-Mb chromosome. Productive bidirectional replication initiated synchronously at both spatially separate origins. Newly replicated DNA from both origins was segregated sequentially as replication progressed, with two temporally and spatially separate replication termination events. Replication initiation occurred at a cell volume identical to that of cells with a single WT origin, showing that initiation control is independent of cellular and chromosomal oriC concentration. Cells containing just the ectopic origin initiated bidirectional replication at the expected cell mass and at the normal cellular location of that region. In all strains, spatial separation of sister loci adjacent to active origins occurred shortly after their replication, independently of whether replication initiated at the normal origin, the ectopic origin, or both origins.

High-copy bacterial plasmids diffuse in the nucleoid-free space, replicate stochastically and are randomly partitioned at cell division

Bacterial plasmids play important roles in the metabolism, pathogenesis and bacterial evolution and are highly versatile biotechnological tools. Stable inheritance of plasmids depends on their autonomous replication and efficient partition to daughter cells at cell division. Active partition systems have not been identified for high-copy number plasmids, and it has been generally believed that they are partitioned randomly at cell division. Nevertheless, direct evidence for the cellular location of replicating and nonreplicating plasmids, and the partition mechanism has been lacking. We used as model pJHCMW1, a plasmid isolated from Klebsiella pneumoniae that includes two β-lactamase and two aminoglycoside resistance genes. Here we report that individual ColE1-type plasmid molecules are mobile and tend to be excluded from the nucleoid, mainly localizing at the cell poles but occasionally moving between poles along the long axis of the cell. As a consequence, at the moment of cell division, most plasmid molecules are located at the poles, resulting in efficient random partition to the daughter cells. Complete replication of individual molecules occurred stochastically and independently in the nucleoid-free space throughout the cell cycle, with a constant probability of initiation per plasmid.