David Dunlap

Supercoiling and denaturation in Gal repressor/heat unstable nucleoid protein (HU)-mediated DNA looping

The overall topology of DNA profoundly influences the regulation of transcription and is determined by DNA flexibility as well as the binding of proteins that induce DNA torsion, distortion, and/or looping. Gal repressor (GalR) is thought to repress transcription from the two promoters of the gal operon of Escherichia coli by forming a DNA loop of ≈40 nm of DNA that encompasses the promoters. Associated evidence of a topological regulatory mechanism of the transcription repression is the requirement for a supercoiled DNA template and the histone-like heat unstable nucleoid protein (HU). By using single-molecule manipulations to generate and finely tune tension in DNA molecules, we directly detected GalR/HU-mediated DNA looping and characterized its kinetics, thermodynamics, and supercoiling dependence. The factors required for gal DNA looping in single-molecule experiments (HU, GalR and DNA supercoiling) correspond exactly to those necessary for gal repression observed both in vitro and in vivo. Our single-molecule experiments revealed that negatively supercoiled DNA, under slight tension, denatured to facilitate GalR/HU-mediated DNA loop formation. Such topological intermediates may operate similarly in other multiprotein complexes of transcription, replication, and recombination.

Calibration of optical tweezers with differential interference contrast signals

A comparison of different calibration methods for optical tweezers with the differential interference contrast (DIC) technique was performed to establish the uses and the advantages of each method. A detailed experimental and theoretical analysis of each method was performed with emphasis on the anisotropy involved in the DIC technique and the noise components in the detection. Finally, a time of flight method that permits the reconstruction of the optical potential well was demonstrated.

FAK+ and PYK2/CAKβ, two related tyrosine kinases highly expressed in the central nervous system: similarities and differences in the expression pattern

Focal adhesion kinase (FAK) and proline‐rich tyrosine kinase 2/cell adhesion kinase β (PYK2/CAKβ) are related, non‐receptor, cytoplasmic tyrosine kinases, highly expressed in the central nervous system (CNS). In addition, FAK+ is a splice isoform of FAK containing a 3‐amino acid insertion in the carboxy‐terminal region. In rat hippocampal slices, FAK+ and PYK2/CAKβ are differentially regulated by neurotransmitters and depolarization. We have studied the regional and cellular distribution of these kinases in adult rat brain and during development. Whereas PYK2/CAKβ expression increased with postnatal age and was maximal in the adult, FAK+ levels were stable. PYK2/CAKβ mRNAs, detected by in situ hybridization, were expressed at low levels in the embryonic brain, and became very abundant in the adult forebrain. Immunocytochemistry of the adult brain showed a widespread neuronal distribution of FAK+ and PYK2/CAKβ immunoreactivities (ir). PYK2/CAKβ appeared to be particularly abundant in the hippocampus. In hippocampal neurons in culture at early stages of development, FAK+ and PYK2/CAKβ were enriched in the perikarya and growth cones. FAK+ extended to the periphery of the growth cones tips, whereas PYK2/CAKβ appeared to be excluded from the lamellipodia. During the establishment of polarity, a proximal‐distal gradient of increasing PYK2/CAKβ‐ir could be observed in the growing axon. In most older neurons, FAK+‐ir was confined to the cell bodies, whereas PYK2/CAKβ‐ir was also present in the processes. In vitro and in vivo, a subpopulation of neurons displayed neurites with intense FAK+‐ir. Thus, FAK+ and PYK2/CAKβ are differentially regulated during development yet they are both abundantly expressed in the adult brain, with distinctive but overlapping distributions.

Sequestration of Toxic Oligomers by HspB1 as a Cytoprotective Mechanism

ABSTRACT Small heat shock proteins (sHsps) are molecular chaperones that protect cells from cytotoxic effects of protein misfolding and aggregation. HspB1, an sHsp commonly associated with senile plaques in Alzheimers disease (AD), prevents the toxic effects of Aβ aggregates in vitro. However, the mechanism of this chaperone activity is poorly understood. Here, we observed that in two distinct transgenic mouse models of AD, mouse HspB1 (Hsp25) localized to the penumbral areas of plaques. We have demonstrated that substoichiometric amounts of human HspB1 (Hsp27) abolish the toxicity of Aβ oligomers on N2a (mouse neuroblastoma) cells. Using biochemical methods, spectroscopy, light scattering, and microscopy methods, we found that HspB1 sequesters toxic Aβ oligomers and converts them into large nontoxic aggregates. HspB1 was overexpressed in N2a cells in response to treatment with Aβ oligomers. Cultured neurons from HspB1-deficient mice were more sensitive to oligomer-mediated toxicity than were those from wild-type mice. Our results suggest that sequestration of oligomers by HspB1 constitutes a novel cytoprotective mechanism of proteostasis. Whether chaperone-mediated cytoprotective sequestration of toxic aggregates may bear clues to plaque deposition and may have potential therapeutic implications must be investigated in the future.

Direct demonstration and quantification of long-range DNA looping by the λ bacteriophage repressor

Recently, it was proposed that DNA looping by the λ repressor (CI protein) strengthens repression of lytic genes during lysogeny and simultaneously ensures efficient switching to lysis. To investigate this hypothesis, tethered particle motion experiments were performed and dynamic CI-mediated looping of single DNA molecules containing the λ repressor binding sites separated by 2317 bp (the wild-type distance) was quantitatively analyzed. DNA containing all three intact operators or with mutated o3 operators were compared. Modeling the thermodynamic data established the free energy of CI octamer-mediated loop formation as 1.7 kcal/mol, which decreased to –0.7 kcal/mol when supplemented by a tetramer (octamer+tetramer-mediated loop). These results support the idea that loops secured by an octamer of CI bound at oL1, oL2, oR1 and oR2 operators must be augmented by a tetramer of CI bound at the oL3 and oR3 to be spontaneous and stable. Thus the o3 sites are critical for loops secured by the CI protein that attenuate cI expression.

Diffusion of light-harvesting complex II in the thylakoid membranes

The light‐harvesting complex II (LHCII) is the main energy absorber for photosynthesis in green plants, and its translocation between photosystems I and II is the primary means of energy redistribution between them. Using single‐particle tracking, we performed the first measurement of the mobility of LHCII in the photosynthetic membranes in both the nonphosphorylated and the phosphorylated (P‐LHCII) conformations. These are part of an important, reversible, energy re‐equilibration process called the state transition. We found that the population of P‐LHCII in unappressed membranes is more mobile than the population of non‐P‐LHCII from the same regions.

Dividing a supercoiled DNA molecule into two independent topological domains

Both prokaryotic and eukaryotic chromosomes are organized into many independent topological domains. These topological domains may be formed through constraining each DNA end from rotating by interacting with nuclear proteins; i.e., DNA-binding proteins. However, so far, evidence to support this hypothesis is still elusive. Here we developed two biochemical methods; i.e., DNA-nicking and DNA-gyrase methods to examine whether certain sequence-specific DNA-binding proteins are capable of separating a supercoiled DNA molecule into distinct topological domains. Our approach is based on the successful construction of a series of plasmid DNA templates that contain many tandem copies of one or two DNA-binding sites in two different locations. With these approaches and atomic force microscopy, we discovered that several sequence-specific DNA-binding proteins; i.e., lac repressor, gal repressor, and λ O protein, are able to divide a supercoiled DNA molecule into two independent topological domains. These topological domains are stable under our experimental conditions. Our results can be explained by a topological barrier model in which nucleoprotein complexes confine DNA supercoils to localized regions. We propose that DNA topological barriers are certain nucleoprotein complexes that contain stable toroidal supercoils assembled from DNA-looping or tightly wrapping DNA around DNA-binding proteins. The DNA topological barrier model may be a general mechanism for certain DNA-binding proteins, such as histone or histone-like proteins, to modulate topology of chromosome DNA in vivo.

Novel tethered particle motion analysis of CI protein-mediated DNA looping in the regulation of bacteriophage lambda

The tethered particle motion (TPM) technique has attracted great interest because of its simplicity and the wealth of information that it can provide on protein-induced conformational changes in nucleic acids. Here we present an approach to TPM methodology and analysis that increases the efficiency of data acquisition and facilitates interpretation of TPM assays. In particular, the statistical analysis that we propose allows fast data processing, minimal data selection and visual display of the distribution of molecular behaviour. The methodology proved useful in verifying CI protein-mediated DNA looping in bacteriophage λ and in differentiating between two different types of loops, stable and dynamic, whose relative occurrence seems to be a function of the distance between the operators as well as their relative angular orientation. Furthermore, the statistical analysis indicates that CI binding per se slightly shortens the DNA.

Quantitation of the DNA tethering effect in long-range DNA looping in vivo and in vitro using the Lac and λ repressors.

Significance Proteins bound to DNA often interact with proteins bound elsewhere on the same DNA to regulate gene expression. The intervening DNA tethers the proteins near each other, making their interaction efficient and specific, but the importance of this tethering effect is poorly understood at large DNA separations. We quantitated tethering inside bacterial cells, using two different proteins at separations up to 10,000 bp, to show that tethering is strong enough to drive efficient interactions over these distances. The same interactions were ∼10-fold weaker outside cells, implying that cellular factors enhance tethering. However, tethering was lost at a DNA separation of 500,000 bp inside bacteria, indicating special mechanisms inside eukaryotic cells to provide efficient and specific interactions over such distances. Efficient and specific interactions between proteins bound to the same DNA molecule can be dependent on the length of the DNA tether that connects them. Measurement of the strength of this DNA tethering effect has been largely confined to short separations between sites, and it is not clear how it contributes to long-range DNA looping interactions, such as occur over separations of tens to hundreds of kilobase pairs in vivo. Here, gene regulation experiments using the LacI and λ CI repressors, combined with mathematical modeling, were used to quantitate DNA tethering inside Escherichia coli cells over the 250- to 10,000-bp range. Although LacI and CI loop DNA in distinct ways, measurements of the tethering effect were very similar for both proteins. Tethering strength decreased with increasing separation, but even at 5- to 10-kb distances, was able to increase contact probability 10- to 20-fold and drive efficient looping. Tethering in vitro with the Lac repressor was measured for the same 600-to 3,200-bp DNAs using tethered particle motion, a single molecule technique, and was 5- to 45-fold weaker than in vivo over this range. Thus, the enhancement of looping seen previously in vivo at separations below 500 bp extends to large separations, underlining the need to understand how in vivo factors aid DNA looping. Our analysis also suggests how efficient and specific looping could be achieved over very long DNA separations, such as what occurs between enhancers and promoters in eukaryotic cells.

Atomic force microscopy study of DNA deposited on poly l‐ornithine‐coated mica

Analyses of individual biomolecules, like DNA, or DNA–protein complexes, via atomic force microscopy, require ‘gentle’ methods to immobilize DNA on surfaces, which allow the ensemble of molecules to adopt conformations dictated primarily by their physical characteristics, and which possibly permit the use of a wide selection of buffers. We show that poly‐l‐ornithine‐coated mica is a good substrate for fast, reliable deposition of DNA for wet or dry imaging. The surface firmly secures DNA, which retains the B‐form helical rise (0.34 nm bp−1). The conformations of DNA that result are reminiscent of three‐dimensional random coils projected on to a plane. The contrast is good, especially in solution, and buffers with physiological concentrations of salt with or without divalent cations may be used. This is important for comparison of scanning probe microscopy results with those obtained by different techniques.