Protamine-Induced Condensation and Decondensation of the Same DNA Molecule
10.1126/science.286.5437.120
Crossref journal-article
American Association for the Advancement of Science (AAAS)
Science (221)
Abstract

The DNA in sperm and certain viruses is condensed by arginine-rich proteins into toroidal subunits, a form of packaging that inactivates their entire genome. Individual DNA molecules were manipulated with an optical trap to examine the kinetics of torus formation induced by the binding of protamine and a subset of its DNA binding domain, Arg 6 . Condensation and decondensation experiments with λ-phage DNA show that toroid formation and stability are influenced by the number of arginine-rich anchoring domains in protamine. The results explain why protamines contain so much arginine and suggest that these proteins must be actively removed from sperm chromatin after fertilization.

Bibliography

Brewer, L. R., Corzett, M., & Balhorn, R. (1999). Protamine-Induced Condensation and Decondensation of the Same DNA Molecule. Science, 286(5437), 120–123.

Authors 3
  1. Laurence R. Brewer (first)
  2. Michele Corzett (additional)
  3. Rod Balhorn (additional)
References 32 Referenced 230
  1. Hud N. V., Allen M. J., Downing K. H., Lee J., Balhorn R., Biochem. Biophys. Res. Commun. 193, 1347 (1993). (10.1006/bbrc.1993.1773) / Biochem. Biophys. Res. Commun. by Hud N. V. (1993)
  2. R. Balhorn et al. in The Male Gamete: From Basic Science to Clinical Applications C. Gagnon Ed. (Cache River Press Vienna IL 1999) pp. 55–70.
  3. Richards K. E., Williams R. C., Calender R., J. Mol. Biol. 78, 255 (1973); (10.1016/0022-2836(73)90114-9) / J. Mol. Biol. by Richards K. E. (1973)
  4. Klimenko S. M. Tikchonenko T. I. Andreev V. M 23 523 (1967); (10.1016/S0022-2836(67)80122-0)
  5. ; N. V. Hud Biophys. J. 69 1355 (1995). (10.1016/S0006-3495(95)80002-0)
  6. R. Balhorn in Molecular Biology of Chromosome Function K. W. Adolph Ed. (Springer-Verlag New York 1989) p. 366. (10.1007/978-1-4612-3652-8_17)
  7. Hud N. V., Milanovich F. P., Balhorn R., Biochemistry 33, 7528 (1994). (10.1021/bi00190a005) / Biochemistry by Hud N. V. (1994)
  8. Bench G. S., Friz A. M., Corzett M. H., Morse D. H., Balhorn R., Cytometry 23, 263 (1996). (10.1002/(SICI)1097-0320(19960401)23:4<263::AID-CYTO1>3.0.CO;2-I) / Cytometry by Bench G. S. (1996)
  9. T. M. Cao M. T. Sung Biochem. Biophys. Res. Commun. 108 (1982); (10.1016/0006-291X(82)92107-6)
  10. Maeda S., Kamita S. G., Kataoka H., Repeat. Virol. 180, 807 (1991). (10.1016/0042-6822(91)90096-T) / Repeat. Virol. by Maeda S. (1991)
  11. Widom J., Baldwin R. L., J. Mol. Biol. 144, 431 (1980); (10.1016/0022-2836(80)90330-7) / J. Mol. Biol. by Widom J. (1980)
  12. Post C. B., Zimm B. H., Biopolymers 21, 2139 (1982); (10.1002/bip.360211105) / Biopolymers by Post C. B. (1982)
  13. Porschke D., J. Mol. Biol. 222, 423 (1991). (10.1016/0022-2836(91)90220-Z) / J. Mol. Biol. by Porschke D. (1991)
  14. Watanabe F., Schwartz G., J. Mol. Biol. 163, 485 (1983). (10.1016/0022-2836(83)90070-0) / J. Mol. Biol. by Watanabe F. (1983)
  15. Arscott P. G., Li A., Bloomfield V. A., Biopolymers 30, 619 (1990); (10.1002/bip.360300514) / Biopolymers by Arscott P. G. (1990)
  16. Bloomfield V. A. 44 269 (1998). (10.1002/(SICI)1097-0282(1997)44:3<269::AID-BIP6>3.0.CO;2-T)
  17. Bustamante C., Annu. Rev. Biophys. Biophys. Chem. 20, 415 (1991). (10.1146/annurev.bb.20.060191.002215) / Annu. Rev. Biophys. Biophys. Chem. by Bustamante C. (1991)
  18. Ueda M., Yoshikowa K., Phys. Rev. Lett. 77, 2133 (1996). (10.1103/PhysRevLett.77.2133) / Phys. Rev. Lett. by Ueda M. (1996)
  19. Baumann C. G., Smith S. B., Bloomfield V. A., Bustamante C., Proc. Natl. Acad. Sci. U.S.A. 94, 6185 (1997). (10.1073/pnas.94.12.6185) / Proc. Natl. Acad. Sci. U.S.A. by Baumann C. G. (1997)
  20. λ-phage DNA molecules were labeled by annealing and ligating a 5′-biotin-labeled 12-base oligonucleotide (Genosys Biotechnologies Inc.) to the cos site. Concatemers of various lengths were formed by annealing unlabeled λ-phage DNA to the biotin-labeled DNA. The biotinylated DNA (2.2 μg) was attached to 1.8 × 10 8 1-μm streptavidin beads (Interfacial Dynamics) in 100 mM NaHCO 3 (pH 8) at 37°C for 1 hour. The DNA-bead preparation was diluted to 0.2 μg/ml with degassed 50% (w/v) sucrose 100 mM NaHCO 3 (pH 8) 30 mM dithiothreitol (DTT) 0.1 μM YOYO-1 (one dye molecule per 4 to 5 bp of DNA) and incubated for 2 hours before use. Salmon protamine (Sigma) was purified by high-pressure liquid chromatography and lyophilized. Arg 6 (SynPeP) was used as provided. The proteins were dissolved in 50% sucrose containing 100 mM NaHCO 3 (pH 8) diluted each day to the required concentrations in degassed 50% sucrose 100 mM NaHCO 3 (pH 8) 30 mM DTT and stored under nitrogen.
  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).
  23. The salmon protamine used in these experiments contains 21 positively charged arginines distributed throughout the length of the protein (32 amino acids).
  24. B. Ostrovsky and Y. Bar-Yam Biophys. J. 68 1694 (1995). (10.1016/S0006-3495(95)80347-4)
  25. Stokes' law f = 6πη rv where η is the buffer viscosity (15.4 cp) r is the sphere radius and v is the buffer velocity (50 μm/s) was used to calculate the frictional force on the fully formed toroid. The toroid used to calculate the frictional force had a 90-nm outer diameter and a 30-nm inner diameter and was 20 nm thick (1).
  26. Because we cannot confirm that every protamine has dissociated from the decondensed DNA molecule these experiments provide only a maximum value for the off-rate.
  27. Ruiz-Lara S. A., Cornudella L., Rodriguez-Campos A., Eur. J. Biochem. 240, 186 (1996). (10.1111/j.1432-1033.1996.0186h.x) / Eur. J. Biochem. by Ruiz-Lara S. A. (1996)
  28. M. Nakano et al. J. Biochem. 105 133 (1989). (10.1093/oxfordjournals.jbchem.a122607)
  29. With an off-rate this high molecules containing a single Arg 6 domain would remain bound to the sperm genome for only 2 hours. This is too short a period of time because the protamine must remain bound to DNA and maintain the sperm chromatin in an inactive state for up to 2 weeks before fertilization.
  30. Perkins T. T., Smith D. E., Larson R. G., Chu S., Science 268, 83 (1995). (10.1126/science.7701345) / Science by Perkins T. T. (1995)
  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.
Dates
Type When
Created 23 years, 1 month ago (July 27, 2002, 5:42 a.m.)
Deposited 1 year, 7 months ago (Jan. 13, 2024, 3:50 a.m.)
Indexed 1 week, 2 days ago (Aug. 27, 2025, 12:15 p.m.)
Issued 25 years, 11 months ago (Oct. 1, 1999)
Published 25 years, 11 months ago (Oct. 1, 1999)
Published Print 25 years, 11 months ago (Oct. 1, 1999)
Funders 0

None

@article{Brewer_1999, title={Protamine-Induced Condensation and Decondensation of the Same DNA Molecule}, volume={286}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.286.5437.120}, DOI={10.1126/science.286.5437.120}, number={5437}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Brewer, Laurence R. and Corzett, Michele and Balhorn, Rod}, year={1999}, month=oct, pages={120–123} }