ABSTRACT
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.
Subject(s)
DNA/chemistry , DNA/metabolism , Protamines/chemistry , Protamines/metabolism , Animals , Bacteriophage lambda/genetics , Binding Sites , Cattle , Chromatin/chemistry , Chromatin/metabolism , Cricetinae , Humans , Male , Mice , Microscopy, Atomic Force , Microscopy, Electron, Transmission , Molecular Conformation , Salmon , Spermatozoa/chemistry , Spermatozoa/metabolismABSTRACT
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.
Subject(s)
DNA/chemistry , DNA/ultrastructure , Microfluidics/methods , Micromanipulation/methods , Models, Chemical , Base Sequence , Computer Simulation , Molecular Sequence Data , Nucleic Acid ConformationABSTRACT
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.
Subject(s)
DNA/chemistry , Nuclear Lamina/metabolism , Proteins/chemistry , Biosensing Techniques , Microfluidic Analytical Techniques , Microfluidics , Microscopy, Atomic Force , Microscopy, Fluorescence/methods , Models, Biological , Protein Binding , Surface PropertiesABSTRACT
Mitochondrial and nuclear DNA are packaged by proteins in a very different manner. Although protein-DNA complexes called "nucleoids" have been identified as the genetic units of mitochondrial inheritance in yeast and man, little is known about their physical structure. The yeast mitochondrial protein Abf2p was shown to be sufficient to compact linear dsDNA, without the benefit of supercoiling, using optical and atomic force microscopy single molecule techniques. The packaging of DNA by Abf2p was observed to be very weak as evidenced by a fast Abf2p off-rate (k(off) = 0.014 +/- 0.001 s(-1)) and the extremely small forces (<0.6 pN) stabilizing the condensed protein-DNA complex. Atomic force microscopy images of individual complexes showed the 190-nm structures are loosely packaged relative to nuclear chromatin. This organization may leave mtDNA accessible for transcription and replication, while making it more vulnerable to damage.
