Mechanisms of DNA Organization Unraveled with Novel Single-Molecule Methods

MacKintosh, F.C. [Promotor]Wuite, G.J.L. [Copromotor

DNA-tension dependence of restriction enzyme activity reveals mechanochemical properties of the reaction pathway

Type II restriction endonucleases protect bacteria against phage infections by cleaving recognition sites on foreign double-stranded DNA (dsDNA) with extraordinary specificity. This capability arises primarily from large conformational changes in enzyme and/or DNA upon target sequence recognition. In order to elucidate the connection between the mechanics and the chemistry of DNA recognition and cleavage, we used a single-molecule approach to measure rate changes in the reaction pathway of EcoRV and BamHI as a function of DNA tension. We show that the induced-fit rate of EcoRV is strongly reduced by such tension. In contrast, BamHI is found to be insensitive, providing evidence that both substrate binding and hydrolysis are not influenced by this force. Based on these results, we propose a mechanochemical model of induced-fit reactions on DNA, allowing determination of induced-fit rates and DNA bend angles. Finally, for both enzymes a strongly decreased association rate is obtained on stretched DNA, presumably due to the absence of intradomain dissociation/re-association between non-specific sites (jumping). The obtained results should apply to many other DNA-associated proteins. © The Author 2005. Published by Oxford University Press. All rights reserved

The architectural role of nucleoid-associated proteins in the organization of bacterial chromatin: A molecular perspective

The bacterial genome is folded into a compact structure called the nucleoid. Considerable compaction of the DNA molecule is required in order to reduce its volume below that of the cell. Several mechanisms, such as molecular crowding and DNA supercoiling contribute to the compactness of the nucleoid. Besides these mechanisms, a number of architectural proteins associate with the chromosomal DNA and cause it to fold into a compact structure by bridging, bending or wrapping DNA. In this review, we provide an overview of the major nucleoid-associated proteins from a structural perspective and we discuss their possible roles in dynamically shaping the bacterial nucleoid. © 2006 Elsevier Inc. All rights reserved

Visualizing the Formation and Collapse of DNA Toroids

In living organisms, DNA is generally confined into very small volumes. In most viruses, positively charged multivalent ions assist the condensation of DNA into tightly packed toroidal structures. Interestingly, such cations can also induce the spontaneous formation of DNA toroids in vitro. To resolve the condensation dynamics and stability of DNA toroids, we use a combination of optical tweezers and fluorescence imaging to visualize in real-time spermine-induced (de)condensation in single DNA molecules. By actively controlling the DNA extension, we are able to follow (de)condensation under tension with high temporal and spatial resolution. We show that both processes occur in a quantized manner, caused by individual DNA loops added onto or removed from a toroidal condensate that is much smaller than previously observed in similar experiments. Finally, we present an analytical model that qualitatively captures the experimentally observed features, including an apparent force plateau

Framework for modelling the cost-effectiveness of systemic interventions aimed to reduce youth delinquency

Background: Many interventions initiated within and financed from the health care sector are not necessarily primarily aimed at improving health. This poses important questions regarding the operationalisation of economic evaluations in such contexts. Aims of the Study: We investigated whether assessing cost-effectiveness using state-of-the-art methods commonly applied in health care evaluations is feasible and meaningful when evaluating interventions aimed at reducing youth delinquency. Methods: A probabilistic Markov model was constructed to create a framework for the assessment of the cost-effectiveness of systemic interventions in delinquent youth. For illustrative purposes, Functional Family Therapy (FFT), a systemic intervention aimed at improving family functioning and, primarily, reducing delinquent activity in youths, was compared to Treatment as Usual (TAU). "Criminal activity free years" (CAFYs) were introduced as central outcome measure. Criminal activity may e.g. be based on police contacts or committed crimes. In absence of extensive data and for illustrative purposes the current study based criminal activity on available literature on recidivism. Furthermore, a literature search was performed to deduce the model's structure and parameters. Results: Common cost-effectiveness methodology could be applied to interventions for youth delinquency. Model characteristics and parameters were derived from literature and ongoing trial data. The model resulted in an estimate of incremental costs/CAFY and included long-term effects. Illustrative model results point towards dominance of FFT compared to TAU. Discussion: Using a probabilistic model and the CAFY outcome measure to assess cost-effectiveness of systemic interventions aimed to reduce delinquency is feasible. However, the model structure is limited to three states and the CAFY measure was defined rather crude. Moreover, as the model parameters are retrieved from literature the model results are illustrative in the absence of empirical data. Implications for Health Care Provision and Use : The current model provides a framework to assess the cost-effectiveness of systemic interventions, while taking into account parameter uncertainty and long-term effectiveness. Implications for Health Policies: The framework of the model could be used to assess the cost-effectiveness of systemic interventions alongside (clinical) trial data. Consequently, it is suitable to inform reimbursement decisions, since the value for money of systemic interventions can be demonstrated using a decision analytic model. Implications for Further Research: Future research could be focussed on testing the current model based on extensive empirical data, improving the outcome measure and finding appropriate values for that outcome