Laurence R. Brewer

Processive translocation and DNA unwinding by individual RecBCD enzyme molecules

RecBCD enzyme is a processive DNA helicase and nuclease that participates in the repair of chromosomal DNA through homologous recombination. We have visualized directly the movement of individual RecBCD enzymes on single molecules of double-stranded DNA (dsDNA). Detection involves the optical trapping of solitary, fluorescently tagged dsDNA molecules that are attached to polystyrene beads, and their visualization by fluorescence microscopy. Both helicase translocation and DNA unwinding are monitored by the displacement of fluorescent dye from the DNA by the enzyme. Here we show that unwinding is both continuous and processive, occurring at a maximum rate of 972 ± 172 base pairs per second (0.30 µm s-1), with as many as 42,300 base pairs of dsDNA unwound by a single RecBCD enzyme molecule. The mean behaviour of the individual RecBCD enzyme molecules corresponds to that observed in bulk solution.

DNA condensation by protamine and arginine-rich peptides: Analysis of toroid stability using single DNA molecules

Both somatic cells and sperm have been shown to take up exogenous DNA, but the frequency of its integration is usually low. Scanning probe microscopy studies of sperm chromatin and synthetic DNA‐protamine complexes indicate that the coiling of DNA into toroidal subunits, a process initiated in the maturing spermatid to prepare its genome for delivery into the egg, can be mimicked by simply adding protamine to DNA in vitro. The increased resistance of DNA‐protamine complexes to nuclease digestion and their structural similarity to native sperm chromatin suggest that the packaging of DNA by protamine might offer a new approach for improving the efficiency of DNA uptake by sperm. Decondensation experiments performed with individual DNA molecules have provided a direct measure of the stability of toroids produced using salmon protamine and smaller arginine‐rich peptides. These experiments show that the arginine content of protamine‐related sequences can have a dramatic effect on their rate of dissociation from DNA. This technique and the information it provides can be used to identify protamine analogs that can be bound to DNA to increase the efficiency of its uptake by sperm and other cells. Mol. Reprod. Dev. 56:230–234, 2000. Published 2000 Wiley‐Liss, Inc.

Laminar flow cells for single-molecule studies of DNA-protein interactions

Microfluidic flow cells are used in single-molecule experiments, enabling measurements to be made with high spatial and temporal resolution. We discuss the fundamental processes affecting flow cell operation and describe the flow cells in use at present for studying the interaction of optically trapped or mechanically isolated, single DNA molecules with proteins. To assist the experimentalist in flow cell selection, we review the construction techniques and materials used to fabricate both single- and multiple-channel flow cells and the advantages of each design for different experiments.

Single molecule studies of DNA-protamine interactions.

Single molecule studies of protamine-DNA interactions have characterized the kinetics of protamine binding to DNA and the morphology of the toroidal subunits that comprise sperm chromatin. The results provided by these studies are reviewed, the advantage of using single molecule techniques is discussed, and the implications of the results to the structure, kinetics of toroid formation, and stability of the DNA-protamine complex are described. New measurements of DNA condensation forces induced by the binding of protamine to DNA are also presented. These forces induce a significant tension in constrained segments of DNA and may contribute to the reduction in volume and shaping of the maturing spermatid cell nucleus.

Deciphering the structure of DNA toroids

Toroids are small donut shaped organizational units within sperm chromatin and viruses containing DNA and protein. Investigators first characterized the dimensions of toroids created in vitro, in viruses and in decondensed sperm chromatin using transmission electron and atomic force microscopy. More recent measurements, performed using cryo-electron microscopy, have allowed experimenters to observe the hexagonal organization of DNA within viruses, and toroids created from DNA and cobalt hexammine. However, it has been difficult to obtain information about the assembly of DNA into a toroid, its structure and the biomechanical forces involved because of the limitations of these techniques. Similarly, biophysical studies of toroids utilizing techniques such as circular dichroism or light scattering are difficult to perform and interpret because toroids created using bulk DNA can aggregate and precipitate out of solution even at very low concentrations. The development of optical and magnetic traps has allowed experimenters to manipulate single DNA molecules within microfluidic, multichannel flow cells and measure the structural changes they undergo as they are transformed into toroids. During the past few years investigators have demonstrated that toroids consist of loops of DNA. They have observed the stepwise incorporation of these loops into a toroid that is not in contact with charged surfaces, which might affect its formation. The condensation of a constrained DNA molecule into a toroid was observed to significantly increase its tension, which reduced the size of the DNA loops that form the toroid. This structural information is important for understanding how genomic DNA is assembled and organized within the sperm cell and viruses. In this perspective we discuss what is known about the structure and formation of toroids, what has been learned recently using single molecule techniques and what remaining questions have the potential to be answered using these emerging technologies.

Highly coherent injection-locked laser diode arrays

Two physically separated laser diode arrays were simultaneously injection-locked to the same master laser. The modulus of their mutual coherence function was measured, and the resulting value 0.96 +/- 0.06 was close to the maximum allowed value of 1. The spatial phase of the two injection-locked outputs did not vary from each other by more than 0.05 wave (lambda/20), apart from a phase difference that grew linearly with position, due to the tilt between the two interfering beams. Requirements for the coherent combination of injection-locked laser diode arrays are discussed.

Genome organization by vertebrate sperm nuclear basic proteins (SNBPs)

DNA is tightly packed in the sperm nucleus through its association with specific chromosomal proteins that are heterogeneous in size and known as sperm nuclear basic proteins (SNBPs). Despite their structural diversity, SNBPs are evolutionarily related and can be classified into three major groups or types: histone (H-type); protamine (P-type), and protamine-like (PL-type). The three types are widespread amongst vertebrates. During spermiogenesis these proteins replace the somatic histones that are present at the onset of spermatogenesis. Mammals exhibit an increased level of complexity as transition proteins (TPs) temporarily bind to DNA before being replaced by protamines in the mature sperm. The proper sequential chromatin remodeling is dependent on global post-translational modifications (PTMs) of the chromosomal proteins involved, including acetylation and phosphorylation. A transient 20nm chromatin fiber that is independent of the SNBP type is often formed. The temporally and spatially organized compaction of chromatin during spermiogenesis may be important for the understanding of post-meiotic events such as gene expression, Huntington’s disease CAG expansion, and the remnant histones that are present in the mature sperm of certain mammals, including humans. Alterations in SNBP composition result in DNA damage and infertility, underscoring the importance of these proteins for male germline genome integrity.

Suppression of beam steering in an injection-locked laser diode array

Experimental measurements were made to demonstrate that the degree of beam steering in an injection‐locked laser diode array is related to the divergence of the master laser beam. For a collimated master laser beam the beam steering was suppressed. The injection‐locked laser diode array beam steers with the master laser wavelength because only a portion of the divergent master laser beam satisfies the round trip mode condition.

Three-dimensional imaging of DNA fragments during electrophoresis using a confocal detector

We have measured the 3D distribution of DNA fragments within an electrophoretic band. The measurements were made using a confocal microscope and a photon counting photomultiplier detector. A DNA sequencing standard was loaded into glass microchannel plates containing polyacrylamide gel. The measurements were made by scanning the plates in three dimensions using a mechanical stage under computer control, while electrophoresis was taking place. We found that the distribution of DNA was the same for all the bands measured in the sequencing ladder with an approximate Gaussian distribution along all three axis. These measurements are important to understand what physical forces shape electrophoretic bands confined by a channel and also as an aid in the design of high throughput DNA sequencers.