Protamine-induced condensation and decondensation of the same DNA molecule

Science

Volumn 286, Issue 5437, 1999, Pages 120-123

  Brewer, Laurence R ^a   Corzett, Michele ^a   Balhorn, Rod ^a  

^a LAWRENCE LIVERMORE NATIONAL LABORATORY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

BACTERIOPHAGE DNA; PROTAMINE;

ARTICLE; BINDING AFFINITY; CHROMATIN CONDENSATION; NONHUMAN; PRIORITY JOURNAL; PROTEIN DNA BINDING; PROTEIN DNA INTERACTION; PROTEIN DOMAIN; PROTEIN STABILITY;

ARGININE; BACTERIOPHAGE LAMBDA; BENZOXAZOLES; DNA, VIRAL; FLUORESCENT DYES; INTERCALATING AGENTS; LASERS; MICROSCOPY, FLUORESCENCE; NUCLEIC ACID CONFORMATION; PROTAMINES; QUINOLINIUM COMPOUNDS;

EID: 0033214448     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.286.5437.120     Document Type: Article

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21. The optical trap was formed by focusing laser light from a solid-state Nd-YLF laser (λ= 1.047 μm, Spectra-Physics) through a Zeiss × 100 Plan Neofluor, oil immersion, infinite conjugate, objective. A Zeiss Axioplan microscope was modified by removing the illumination optics to allow entry of the laser. The typical laser power used to form the trap (exiting the microscope objective) was 50 mW.
22. The fluorescence was detected with an image-intensified charge-coupled device camera (PAULTEK), the signal was recorded on an SVHS VCR, and an Argus Image Processor (Hamamatsu) and a frame grabber (National Instruments PCI 1402) were used to perform background subtraction. The lengths of the DNA molecules, measured between the center of the 1-μm bead and the free end of the molecule, were obtained from frame-grabbed images. The length of stained DNA (48.5 kb) extended by flow was measured to be 19.2 ± 0.82 μm at a flow rate of 72 μm/s in 50% sucrose. Under these conditions, DNA is 93% extended (24).
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31. The flow cell contains two channels, each pumped at the same speed with a single syringe pump. The depth of the flow cell was 40 μm and the molecule was typically held 20 μm beneath the coverslip. Flow velocities were maintained at ∼50 μm/s. Using a computer-controlled stage with 0.1-μm resolution to manipulate the position of the flow cell relative to the optical trap, we moved the DNA molecule to the protein side of the flow cell to initiate condensation.
32. Work was performed at Lawrence Livermore National Laboratory (LLNL) under the auspices of the U.S. Department of Energy under contract W-7405-ENG-48. Funding was provided by a LLNL Labwide Laboratory Directed Research Development Award. We thank J. Holzrichter, M. Colvin, and M. Cosman for their suggestions, support, and encouragement; J. W. Cosman and C. Barry for help during the early stages of the study; and J. T. Cosman for generating the computer graphics image of DNA.