Heteroplasmy in action: tracking mtDNA segregation dynamics.

Difficulties in tracking the dynamics of heteroplasmy, a state when a single cell contains distinct mtDNA genotypes, have left our understanding of factors influencing it incomplete. New work now provides a powerful approach for monitoring heteroplasmic yeast populations in real-time, and reveals insights into the forces underlying mtDNA variant segregation.

Chromosome organization shapes replisome dynamics in Caulobacter crescentus.

DNA replication in bacteria takes place on highly compacted chromosomes, where segregation, transcription, and repair must occur simultaneously. Within this dynamic environment, colocalization of sister replisomes has been observed in many bacterial species, driving the hypothesis that a physical linker may tether them together. However, replisome splitting has also been reported in many of the same species, leaving the principles behind replisome organization a long-standing puzzle.

Widespread prevalence of a methylation-dependent switch to activate an essential DNA damage response in bacteria.

DNA methylation plays central roles in diverse cellular processes, ranging from error-correction during replication to regulation of bacterial defense mechanisms. Nevertheless, certain aberrant methylation modifications can have lethal consequences. The mechanisms by which bacteria detect and respond to such damage remain incompletely understood.

SOS-independent bacterial DNA damage responses: diverse mechanisms, unifying function.

Cells across domains of life have dedicated pathways to sense and respond to DNA damage. These responses are broadly termed as DNA damage responses (DDRs). In bacteria, the best studied DDR is the Save our Soul (SOS) response.

Mechanistic insight into the repair of C8-linked pyrrolobenzodiazepine monomer-mediated DNA damage.

Pyrrolobenzodiazepines (PBDs) are naturally occurring DNA binding compounds that possess anti-tumor and anti-bacterial activity. Chemical modifications of PBDs can result in improved DNA binding, sequence specificity and enhanced efficacy. More recently, synthetic PBD monomers have shown promise as payloads for antibody drug conjugates and anti-bacterial agents.

SMC protein RecN drives RecA filament translocation for in vivo homology search.

While the molecular repertoire of the homologous recombination pathways is well studied, the search mechanism that enables recombination between distant homologous regions is poorly understood. Earlier work suggests that the recombinase RecA, an essential component for homology search, forms an elongated filament, nucleating at the break site. How this RecA structure carries out long-distance search remains unclear.

DarT-mediated mtDNA damage induces dynamic reorganization and selective segregation of mitochondria.

Mitochondria are dynamic organelles that play essential roles in cell growth and survival. Processes of fission and fusion are critical for the distribution, segregation, and maintenance of mitochondria and their genomes (mtDNA). While recent work has revealed the significance of mitochondrial organization for mtDNA maintenance, the impact of mtDNA perturbations on mitochondrial dynamics remains less understood.

Fluorescence Recovery After Photobleaching (FRAP) to Study Dynamics of the Structural Maintenance of Chromosome (SMC) Complex in Live Escherichia coli Bacteria.

MukBEF, a structural maintenance of chromosome (SMC) complex, is an important molecular machine for chromosome organization and segregation in Escherichia coli. Fluorescently tagged MukBEF forms distinct spots (or "foci") composed of molecular assemblies in the cell, where it is thought to carry out most of its chromosome-associated activities. Here, we outline the technique of fluorescence recovery after photobleaching (FRAP) as a method to study the properties of YFP-tagged MukB in fluorescent foci.

A CTP-dependent gating mechanism enables ParB spreading on DNA.

Proper chromosome segregation is essential in all living organisms. The ParA-ParB- system is widely employed for chromosome segregation in bacteria. Previously, we showed that ParB requires cytidine triphosphate to escape the nucleation site and spread by sliding to the neighboring DNA (Jalal et al.

Coordination between nucleotide excision repair and specialized polymerase DnaE2 action enables DNA damage survival in non-replicating bacteria.

Translesion synthesis (TLS) is a highly conserved mutagenic DNA lesion tolerance pathway, which employs specialized, low-fidelity DNA polymerases to synthesize across lesions. Current models suggest that activity of these polymerases is predominantly associated with ongoing replication, functioning either at or behind the replication fork. Here we provide evidence for DNA damage-dependent function of a specialized polymerase, DnaE2, in replication-independent conditions.

Asymmetric chromosome segregation and cell division in DNA damage-induced bacterial filaments.

Faithful propagation of life requires coordination of DNA replication and segregation with cell growth and division. In bacteria, this results in cell size homeostasis and periodicity in replication and division. The situation is perturbed under stress such as DNA damage, which induces filamentation as cell cycle progression is blocked to allow for repair.

Evolutionary and Comparative Analysis of Bacterial Nonhomologous End Joining Repair.

DNA double-strand breaks (DSBs) are a threat to genome stability. In all domains of life, DSBs are faithfully fixed via homologous recombination. Recombination requires the presence of an uncut copy of duplex DNA which is used as a template for repair.

Visualizing mutagenic repair: novel insights into bacterial translesion synthesis.

DNA repair is essential for cell survival. In all domains of life, error-prone and error-free repair pathways ensure maintenance of genome integrity under stress. Mutagenic, low-fidelity repair mechanisms help avoid potential lethality associated with unrepaired damage, thus making them important for genome maintenance and, in some cases, the preferred mode of repair.

Live-Cell Fluorescence Imaging of RecN in Caulobacter crescentus Under DNA Damage.

Structural maintenance of chromosomes (SMC) proteins play a central role in the organization, segregation and maintenance of chromosomes across domains of life. In bacteria, an SMC-family protein, RecN, has been implicated to have important functions in DNA damage repair. Recent studies have suggested that RecN is required to increase chromosome cohesion in response to DNA damage and may also stimulate specific events during recombination-based repair.

Tracking Bacterial Chromosome Dynamics with Microfluidics-Based Live Cell Imaging.

In bacteria, chromosomes are highly organized within the limited volume of the cell to form a nucleoid. Recent application of microscopy and chromosome conformation capture techniques have together provided a comprehensive understanding of the nature of this organization and the role of factors such as the structural maintenance of chromosomes (SMC) proteins in the establishment and maintenance of the same. In this chapter, we outline a microfluidics-based approach for live cell imaging of Escherichia coli chromosome dynamics in wild-type cells.

Global analysis of double-strand break processing reveals in vivo properties of the helicase-nuclease complex AddAB.

In bacteria, double-strand break (DSB) repair via homologous recombination is thought to be initiated through the bi-directional degradation and resection of DNA ends by a helicase-nuclease complex such as AddAB. The activity of AddAB has been well-studied in vitro, with translocation speeds between 400-2000 bp/s on linear DNA suggesting that a large section of DNA around a break site is processed for repair. However, the translocation rate and activity of AddAB in vivo is not known, and how AddAB is regulated to prevent excessive DNA degradation around a break site is unclear.

Using Fluorescence Recovery After Photobleaching (FRAP) to Study Dynamics of the Structural Maintenance of Chromosome (SMC) Complex In Vivo.

The SMC complex, MukBEF, is important for chromosome organization and segregation in Escherichia coli. Fluorescently tagged MukBEF forms distinct spots (or "foci") in the cell, where it is thought to carry out most of its chromosome associated activities. This chapter outlines the technique of Fluorescence Recovery After Photobleaching (FRAP) as a method to study the properties of YFP-tagged MukB in fluorescent foci.

MatP regulates the coordinated action of topoisomerase IV and MukBEF in chromosome segregation.

The Escherichia coli SMC complex, MukBEF, forms clusters of molecules that interact with the decatenase topisomerase IV and which are normally associated with the chromosome replication origin region (ori). Here we demonstrate an additional ATP-hydrolysis-dependent association of MukBEF with the replication termination region (ter). Consistent with this, MukBEF interacts with MatP, which binds matS sites in ter.

Bacterial chromosome organization and segregation.

If fully stretched out, a typical bacterial chromosome would be nearly 1 mm long, approximately 1,000 times the length of a cell. Not only must cells massively compact their genetic material, but they must also organize their DNA in a manner that is compatible with a range of cellular processes, including DNA replication, DNA repair, homologous recombination, and horizontal gene transfer. Recent work, driven in part by technological advances, has begun to reveal the general principles of chromosome organization in bacteria.

Rapid pairing and resegregation of distant homologous loci enables double-strand break repair in bacteria.

Double-strand breaks (DSBs) can lead to the loss of genetic information and cell death. Although DSB repair via homologous recombination has been well characterized, the spatial organization of this process inside cells remains poorly understood, and the mechanisms used for chromosome resegregation after repair are unclear. In this paper, we introduced site-specific DSBs in Caulobacter crescentus and then used time-lapse microscopy to visualize the ensuing chromosome dynamics.

The SMC complex MukBEF recruits topoisomerase IV to the origin of replication region in live Escherichia coli.

Unlabelled: The Escherichia coli structural maintenance of chromosome (SMC) complex, MukBEF, and topoisomerase IV (TopoIV) interact in vitro through a direct contact between the MukB dimerization hinge and the C-terminal domain of ParC, the catalytic subunit of TopoIV. The interaction stimulates catalysis by TopoIV in vitro. Using live-cell quantitative imaging, we show that MukBEF directs TopoIV to ori, with fluorescent fusions of ParC and ParE both forming cellular foci that colocalize with those formed by MukBEF throughout the cell cycle and in cells unable to initiate DNA replication.

In vivo architecture and action of bacterial structural maintenance of chromosome proteins.

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.

The Escherichia coli SMC complex, MukBEF, shapes nucleoid organization independently of DNA replication.

SMC (structural maintenance of chromosomes) complexes function ubiquitously in organizing and maintaining chromosomes. Functional fluorescent derivatives of the Escherichia coli SMC complex, MukBEF, form foci that associate with the replication origin region (ori). MukBEF impairment results in mispositioning of ori and other loci in steady-state cells.