Probing protein heterogeneity in the plasma membrane using PALM and pair correlation analysis.

Photoactivated localization microscopy (PALM) is a powerful approach for investigating protein organization, yet tools for quantitative, spatial analysis of PALM datasets are largely missing. Combining pair-correlation analysis with PALM (PC-PALM), we provide a method to analyze complex patterns of protein organization across the plasma membrane without determination of absolute protein numbers. The approach uses an algorithm to distinguish a single protein with multiple appearances from clusters of proteins.

Barrier-to-autointegration factor (BAF) condenses DNA by looping.

Barrier-to-autointegration factor (BAF) is a protein that has been proposed to compact retroviral DNA, making it inaccessible as a target for self-destructive integration into itself (autointegration). BAF also plays an important role in nuclear organization. We studied the mechanism of DNA condensation by BAF using total internal reflection fluorescence microscopy.

Micromanipulation studies of chromatin fibers in Xenopus egg extracts reveal ATP-dependent chromatin assembly dynamics.

We have studied assembly of chromatin using Xenopus egg extracts and single DNA molecules held at constant tension by using magnetic tweezers. In the absence of ATP, interphase extracts were able to assemble chromatin against DNA tensions of up to 3.5 piconewtons (pN).

Tn5 transposase loops DNA in the absence of Tn5 transposon end sequences.

Transposases mediate transposition first by binding specific DNA end sequences that define a transposable element and then by organizing protein and DNA into a highly structured and stable nucleoprotein 'synaptic' complex. Synaptic complex assembly is a central checkpoint in many transposition mechanisms. The Tn5 synaptic complex contains two Tn5 transposase subunits and two Tn5 transposon end sequences, exhibits extensive protein-end sequence DNA contacts and is the node of a DNA loop.

Mechanism of chromosome compaction and looping by the Escherichia coli nucleoid protein Fis.

Fis, the most abundant DNA-binding protein in Escherichia coli during rapid growth, has been suspected to play an important role in defining nucleoid structure. Using bulk-phase and single-DNA molecule experiments, we analyze the structural consequences of non-specific binding by Fis to DNA. Fis binds DNA in a largely sequence-neutral fashion at nanomolar concentrations, resulting in mild compaction under applied force due to DNA bending.

Low-force DNA condensation and discontinuous high-force decondensation reveal a loop-stabilizing function of the protein Fis.

We report single-DNA-stretching experiments showing that the protein Fis, an abundant bacterial chromosome protein of E. coli, mediates a dramatic DNA condensation to zero length. This condensation occurs abruptly when DNA tension is reduced below a protein-concentration-dependent threshold f* < 1 pN.

Defining a centromere-like element in Bacillus subtilis by Identifying the binding sites for the chromosome-anchoring protein RacA.

Chromosome segregation during sporulation in Bacillus subtilis involves the anchoring of sister chromosomes to opposite ends of the cell. Anchoring is mediated by RacA, which acts as a bridge between a centromere-like element in the vicinity of the origin of replication and the cell pole. To define this element we mapped RacA binding sites by performing chromatin immunoprecipitation in conjunction with gene microarray analysis.

Micromechanical analysis of the binding of DNA-bending proteins HMGB1, NHP6A, and HU reveals their ability to form highly stable DNA-protein complexes.

The mechanical response generated by binding of the nonspecific DNA-bending proteins HMGB1, NHP6A, and HU to single tethered 48.5 kb lambda-DNA molecules is investigated using DNA micromanipulation. As protein concentration is increased, the force needed to extend the DNA molecule increases, due to its compaction by protein-generated bending.

Near-field-magnetic-tweezer manipulation of single DNA molecules.

We have developed an instrument for micromanipulation of single DNA molecules end labeled with 3-microm-diameter paramagnetic particles. A small, permanent magnet that can be moved as close as 10 microm to the particle being manipulated can generate forces in excess of 200 pN, significantly larger than obtained in other recent "magnetic-tweezer" studies. Our instrument generates these forces in the focal plane of a microscope objective, allowing straightforward real-time observation of molecule extension with a position resolution of approximately 30 nm.