Single-molecule studies of mechanical and helicase-catalyzed disruption of nucleic acid duplexes

Nucleic acids (e.g. DNA, RNA) are subjected to numerous twisting, bending, and stretching forces within cells, and enzymes process them in a variety of ways. The behavior of nucleic acids in response to applied forces and enzymatic activity is therefore necessary for a fundamental understanding of biology. Furthermore, a detailed knowledge of nucleic acids and the enzymes that process them has fueled advances in bio- and nano-technology. In this thesis, we focus on two main systems: the elastic behavior of ultrashort nucleic acids and the activity of E. coli UvrD helicase. First, we use a hybrid instrument combining high-resolution optical tweezers with single-fluorophore sensitivity to observe the hybridization of ultrashort (<15 nt) DNA and RNA oligonucleotides under tension, one molecule at a time. We quantify the effect of tension on the rates of hybridization, and in doing so determine the elastic behavior of the transition state for the reaction. We then investigate the elasticity of the ultrashort oligonucleotides by observing the change in extension that takes place during hybridization. Our results enable us to produce a model describing the shear-induced fraying of base-pairs in a nucleic acid duplex. We then use similar single-molecule techniques to characterize E. coli UvrD helicase. First, we investigate UvrD’s stepping dynamics by directly observing individual motor steps of the protein. Then, we examine the factors influencing the ability of UvrD to switch between unzipping and re-zipping behaviors. Finally, we place UvrD in its biological context by observing the effect of its interactions with an accessory protein in DNA mismatch repair

Kinetic and structural mechanism for DNA unwinding by a non-hexameric helicase

UvrD, a model for non-hexameric Superfamily 1 helicases, utilizes ATP hydrolysis to translocate stepwise along single-stranded DNA and unwind the duplex. Previous estimates of its step size have been indirect, and a consensus on its stepping mechanism is lacking. To dissect the mechanism underlying DNA unwinding, we use optical tweezers to measure directly the stepping behavior of UvrD as it processes a DNA hairpin and show that UvrD exhibits a variable step size averaging ~3 base pairs. Analyzing stepping kinetics across ATP reveals the type and number of catalytic events that occur with different step sizes. These single-molecule data reveal a mechanism in which UvrD moves one base pair at a time but sequesters the nascent single strands, releasing them non-uniformly after a variable number of catalytic cycles. Molecular dynamics simulations point to a structural basis for this behavior, identifying the protein-DNA interactions responsible for strand sequestration. Based on structural and sequence alignment data, we propose that this stepping mechanism may be conserved among other non-hexameric helicases

Peptidoglycan synthesis drives a single population of septal cell wall synthases during division in Bacillus subtilis

Bacterial cell division requires septal peptidoglycan (sPG) synthesis by the divisome complex. Treadmilling of the essential tubulin homologue FtsZ has been implicated in septal constriction, though its precise role remains unclear. Here we used live-cell single-molecule imaging of the divisome transpeptidase PBP2B to investigate sPG synthesis dynamics in Bacillus subtilis. In contrast to previous models, we observed a single population of processively moving PBP2B molecules whose motion is driven by peptidoglycan synthesis and is not associated with FtsZ treadmilling. However, despite the asynchronous motions of PBP2B and FtsZ, a partial dependence of PBP2B processivity on FtsZ treadmilling was observed. Additionally, through single-molecule counting experiments we provide evidence that the divisome synthesis complex is multimeric. Our results support a model for B. subtilis division where a multimeric synthesis complex follows a single track dependent on sPG synthesis whose activity and dynamics are asynchronous with FtsZ treadmilling

Can processes make relationships work? The Triple Helix between structure and action

This contribution seeks to explore how complex adaptive theory can be applied at the conceptual level to unpack Triple Helix models. We use two cases to examine this issue – the Finnish Strategic Centres for Science, Technology & Innovation (SHOKs) and the Canadian Business-led Networks of Centres of Excellence (BL-NCE). Both types of centres are organisational structures that aspire to be business-led, with a considerable portion of their activities driven by (industrial) users’ interests and requirements. Reflecting on the centres’ activities along three dimensions – knowledge generation, consensus building and innovation – we contend that conceptualising the Triple Helix from a process perspective will improve the dialogue between stakeholders and shareholders

Experts' Judgments of Management Journal Quality:An Identity Concerns Model

Many lists that purport to gauge the quality of journals in management and organization studies (MOS) are based on the judgments of experts in the field. This article develops an identity concerns model (ICM) that suggests that such judgments are likely to be shaped by the personal and social identities of evaluators. The model was tested in a study in which 168 editorial board members rated 44 MOS journals. In line with the ICM, respondents rated journal quality more highly to the extent that a given journal reflected their personal concerns (associated with having published more articles in that journal) and the concerns of a relevant ingroup (associated with membership of the journal’s editorial board or a particular disciplinary or geographical background). However, judges’ ratings of journals in which they had published were more favorable when those journals had a low-quality reputation, and their ratings of journals that reflected their geographical and disciplinary affiliations were more favorable when those journals had a high-quality reputation. The findings are thus consistent with the view that identity concerns come to the fore in journal ratings when there is either a need to protect against personal identity threat or a meaningful opportunity to promote social identity

Exhaled Aerosol Transmission of Pandemic and Seasonal H1N1 Influenza Viruses in the Ferret

Person-to-person transmission of influenza viruses occurs by contact (direct and fomites) and non-contact (droplet and small particle aerosol) routes, but the quantitative dynamics and relative contributions of these routes are incompletely understood. The transmissibility of influenza strains estimated from secondary attack rates in closed human populations is confounded by large variations in population susceptibilities. An experimental method to phenotype strains for transmissibility in an animal model could provide relative efficiencies of transmission. We developed an experimental method to detect exhaled viral aerosol transmission between unanesthetized infected and susceptible ferrets, measured aerosol particle size and number, and quantified the viral genomic RNA in the exhaled aerosol. During brief 3-hour exposures to exhaled viral aerosols in airflow-controlled chambers, three strains of pandemic 2009 H1N1 strains were frequently transmitted to susceptible ferrets. In contrast one seasonal H1N1 strain was not transmitted in spite of higher levels of viral RNA in the exhaled aerosol. Among three pandemic strains, the two strains causing weight loss and illness in the intranasally infected ‘donor’ ferrets were transmitted less efficiently from the donor than the strain causing no detectable illness, suggesting that the mucosal inflammatory response may attenuate viable exhaled virus. Although exhaled viral RNA remained constant, transmission efficiency diminished from day 1 to day 5 after donor infection. Thus, aerosol transmission between ferrets may be dependent on at least four characteristics of virus-host relationships including the level of exhaled virus, infectious particle size, mucosal inflammation, and viral replication efficiency in susceptible mucosa

Emergence of a Small-World Functional Network in Cultured Neurons

The functional networks of cultured neurons exhibit complex network properties similar to those found in vivo. Starting from random seeding, cultures undergo significant reorganization during the initial period in vitro, yet despite providing an ideal platform for observing developmental changes in neuronal connectivity, little is known about how a complex functional network evolves from isolated neurons. In the present study, evolution of functional connectivity was estimated from correlations of spontaneous activity. Network properties were quantified using complex measures from graph theory and used to compare cultures at different stages of development during the first 5 weeks in vitro. Networks obtained from young cultures (14 days in vitro) exhibited a random topology, which evolved to a small-world topology during maturation. The topology change was accompanied by an increased presence of highly connected areas (hubs) and network efficiency increased with age. The small-world topology balances integration of network areas with segregation of specialized processing units. The emergence of such network structure in cultured neurons, despite a lack of external input, points to complex intrinsic biological mechanisms. Moreover, the functional network of cultures at mature ages is efficient and highly suited to complex processing tasks

Clinical Sequencing Exploratory Research Consortium: Accelerating Evidence-Based Practice of Genomic Medicine

Despite rapid technical progress and demonstrable effectiveness for some types of diagnosis and therapy, much remains to be learned about clinical genome and exome sequencing (CGES) and its role within the practice of medicine. The Clinical Sequencing Exploratory Research (CSER) consortium includes 18 extramural research projects, one National Human Genome Research Institute (NHGRI) intramural project, and a coordinating center funded by the NHGRI and National Cancer Institute. The consortium is exploring analytic and clinical validity and utility, as well as the ethical, legal, and social implications of sequencing via multidisciplinary approaches; it has thus far recruited 5,577 participants across a spectrum of symptomatic and healthy children and adults by utilizing both germline and cancer sequencing. The CSER consortium is analyzing data and creating publically available procedures and tools related to participant preferences and consent, variant classification, disclosure and management of primary and secondary findings, health outcomes, and integration with electronic health records. Future research directions will refine measures of clinical utility of CGES in both germline and somatic testing, evaluate the use of CGES for screening in healthy individuals, explore the penetrance of pathogenic variants through extensive phenotyping, reduce discordances in public databases of genes and variants, examine social and ethnic disparities in the provision of genomics services, explore regulatory issues, and estimate the value and downstream costs of sequencing. The CSER consortium has established a shared community of research sites by using diverse approaches to pursue the evidence-based development of best practices in genomic medicine

High-resolution imaging of bacterial spatial organization with vertical cell imaging by nanostructured immobilization (VerCINI)

Light microscopy is indispensable for analysis of bacterial spatial organization, yet the sizes and shapes of bacterial cells pose unique challenges to imaging. Bacterial cells are not much larger than the diffraction limit of visible light, and many species have cylindrical shapes and so lie flat on microscope coverslips, yielding low-resolution images when observing their short axes. In this protocol, we describe a pair of recently developed methods named VerCINI (vertical cell imaging by nanostructured immobilization) and µVerCINI (microfluidic VerCINI) that greatly increase spatial resolution and image quality for microscopy of the short axes of bacteria. The concept behind both methods is that cells are imaged while confined vertically inside cell traps made from a nanofabricated mold. The mold is a patterned silicon wafer produced in a cleanroom facility using electron-beam lithography and deep reactive ion etching, which takes ~3 h for fabrication and ~12 h for surface passivation. After obtaining a mold, the entire process of making cell traps, imaging cells and processing images can take ~2–12 h, depending on the experiment. VerCINI and µVerCINI are ideal for imaging any process along the short axes of bacterial cells, as they provide high-resolution images without any special requirements for fluorophores or imaging modalities, and can readily be combined with other imaging methods (e.g., STORM). VerCINI can easily be incorporated into existing projects by researchers with expertise in bacteriology and microscopy. Nanofabrication can be either done in-house, requiring specialist facilities, or outsourced based on this protocol