DNA, Flexibly Flexible

AbstractInvestigators have constructed dsDNA molecules with several different base modifications and have characterized their bending and twisting flexibilities using atomic force microscopy, DNA ring closure, and single-molecule force spectroscopy with optical tweezers. The three methods provide persistence length measurements that agree semiquantitatively, and they show that the persistence length is surprisingly similar for all of the modified DNAs. The circular dichroism spectra of modified DNAs differ substantially. Simple explanations based on base stacking strength, polymer charge, or groove occupancy by functional groups cannot explain the results, which will guide further high-resolution theory and experiments

Tethered Particle Motion Reveals that LacI·DNA Loops Coexist with a Competitor-Resistant but Apparently Unlooped Conformation

AbstractThe lac repressor protein (LacI) efficiently represses transcription of the lac operon in Escherichia coli by binding to two distant operator sites on the bacterial DNA and causing the intervening DNA to form a loop. We employed single-molecule tethered particle motion to observe LacI-mediated loop formation and breakdown in DNA constructs that incorporate optimized operator binding sites and intrinsic curvature favorable to loop formation. Previous bulk competition assays indirectly measured the loop lifetimes in these optimized DNA constructs as being on the order of days; however, we measured these same lifetimes to be on the order of minutes for both looped and unlooped states. In a range of single-molecule DNA competition experiments, we found that the resistance of the LacI-DNA complex to competitive binding is a function of both the operator strength and the interoperator sequence. To explain these findings, we present what we believe to be a new kinetic model of loop formation and DNA competition. In this proposed new model, we hypothesize a new unlooped state in which the unbound DNA-binding domain of the LacI protein interacts nonspecifically with nonoperator DNA adjacent to the operator site at which the second LacI DNA-binding domain is bound

The Structure of Critical Care Transfer Networks

Rationale: Moving patients from low-performing hospitals to high-performing hospitals may improve patient outcomes. These transfers may be particularly important in critical care, where small relative improvements can yield substantial absolute changes in survival. Objective: To characterize the existing critical care network in terms of the pattern of transfers. Methods: In a retrospective cohort study, the nationwide 2005 Medicare fee-for-service claims were used to identify the interhospital transfer of critically ill patients, defined as instances where patients used critical care services in 2 temporally adjacent hospitalizations. Measurements: We measured the characteristics of the interhospital transfer network and the extent to which intensive care unit patients are referred to each hospital in that network--a continuous quantitative measure at the hospital-level known as centrality. We evaluated associations between hospital centrality and organizational, medical, surgical, and radiologic capabilities. Results: There were 47,820 transfers of critically ill patients among 3308 hospitals. 4.5% of all critical care stays of any length involved an interhospital critical care transfer. Hospitals transferred out to a mean of 4.4 other hospitals. More central hospital positions were associated with multiple indicators of increased capability. Hospital characteristics explained 40.7% of the variance in hospitals' centrality. Conclusions: Critical care transfers are common, and traverse an informal but structured network. The centrality of a hospital is associated with increased capability in delivery of services, suggesting that existing transfers generally direct patients toward better resourced hospitals. Studies of this network promise further improvements in patient outcomes and efficiency of care. * Note, if you find this of interest, you may also be interested in the follow-up manuscript exploring the determinants and efficiency of the network in targeting transfers for patients with acute myocardial infarction (AMI, aka heart attacks) at http://hdl.handle.net/2027.42/78005Supported in part by NIH grants HL07891-09 and K08 HL09 1249 and an ATS Fellows Career Development AwardPeer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63027/1/09.Medical.Care.Network.Structure.pd

Flexibility and Control of Protein-DNA Loops

Combined research report and review article on our work in DNA looping. Presented at the Asia and Pacific Workshop on Biological Physics, Singapore, July 2006.Protein-DNA loops are essential for efficient transcriptional repression and activation. The geometry and stability of the archetypal Lac repressor tetramer (LacI)-DNA loop were investigated using designed hyperstable loops containing lac operators bracketing a sequence-directed bend. Electrophoretic mobility shift assays, DNA cyclization, and bulk and single-molecule fluorescence resonance energy transfer (FRET) demonstrate that the DNA sequence controls whether the LacI-DNA loop forms a compact loop with positive writhe or an open loop with little writhe. Monte Carlo methods for simulation of DNA ring closure were extended to DNA loops, including treatment of variable protein hinge angles. The observed distribution of topoisomer products upon cyclization provides a strong constraint on possible models. The experiments and modeling imply that LacI-DNA can adopt a wide range of geometries but has a strong intrinsic preference for an open form. The flexibility of LacI helps explain in vivo observations that DNA looping is less sensitive to DNA length and shape than would be expected from the physical properties of DNA. While DNA cyclization suggests two pools of precursor loops for the 9C14 construct, single-molecule FRET demonstrates a single population. This discrepancy suggests that the LacI-DNA structure is strongly influenced by flanking DNA.NSF, NI

3D reconstruction and comparison of shapes of DNA minicircles observed by cryo-electron microscopy

We use cryo-electron microscopy to compare 3D shapes of 158 bp long DNA minicircles that differ only in the sequence within an 18 bp block containing either a TATA box or a catabolite activator protein binding site. We present a sorting algorithm that correlates the reconstructed shapes and groups them into distinct categories. We conclude that the presence of the TATA box sequence, which is believed to be easily bent, does not significantly affect the observed shapes