Click on Picture for Bio	Sanford Leuba, Ph.D. Cancer Scholar Physical Molecular Biology leuba@nih.gov	Atomic Force Microscopy of Chromatin Fiber

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Molecular biologists have come to realize that chromatin structure may be of major importance in regulating transcription, replication, and repair. Our interests are to understand at the single fiber level, the structure, function, and dynamics of chromatin. To this end, we are applying atomic force microscopy (AFM) to native or reconstituted chromatin fibers with different protein compositions.

The atomic force microscope has resolution on the general order of the electron microscope; however, it also has the ability to work with samples completely immersed in solution at room temperature. Hence, it should be possible to follow biochemical reactions at the single fiber level in real time. Additionally, as the microscope produces the image from a mechanical interaction between the tip and the sample, it is also possible to investigate the mechanical properties and molecular interactions of complex polymers such as chromatin.

Recent Findings and Activities

Imaging of Chromatin Fibers at Low Ionic-Strength & Mathematical Modeling

• Chromatin fibers exhibit irregular, three-dimensional morphology at low-ionic strength.
• Chicken erythrocyte chromatin fibers stripped of linker histones H1 and H5 exhibit a "beads-on-a-string" morphology.
• Modeling of the irregular, three-dimensional arrangement of nucleosomes in the chromatin fiber.
• Modeling of the effect of the removal of linker histone from chromatin fibers.
• Quantitative Measurements by AFM

Chromatin Fiber Compaction With Salt

• NaCl-induced compaction of the extended chromatin fiber, as visualized by AFM imaging.
• MgCl2-induced compaction of the extended chromatin fiber, as visualized by AFM imaging.

Assessment of Contributions of Different Histone Classes to Chromatin Fiber Structure (Anonymous peer-review of Leuba et al., 1998a,b )

• Mild trypsin hydrolysis to analyze the contributions of the linker histone domains and the N-termini of core histone H3 to chromatin fiber structure.
• Reconstitution to selectively determine the contributions of the linker histone domains and the N-termini of core histone H3 to chromatin fiber structure.
  • Reconstitution of linker histone domains onto intact core histone octamer chromatin.
  • Reconstitution of linker histone domains onto H3-tailless chromatin.
  • Schematic presentation of reconstitution results.
  • How our results mesh with the crystal structure of the nucleosome.

  Publications

  • Leuba, S. H., Zlatanova, J. Biology at the single-molecule level Pergamon, Amsterdam, 2001. ISBN: 0-08-044031-2
  • Leuba, S. H., Zlatanova, J., Karymov, M. A., Bash R., Liu, Y.Z. Lohr, D.E. Harrington R.E., and Lindsay, S.M. The mechanical properties of single chromatin fibers under tension. Single Molecules 1 185-192, 2000
  • Zlatanova, J., Lindsay, S. M., and Leuba, S. H. Single molecule force spectroscopy in biology using the atomic force microscope. Prog. Biophys. Mol. Biol. 74 37-61, 2001
  • Leuba, S. H., Zlatanova, J. Single-molecule biochemistry coming of age. Prog. Biophys. Mol. Biol. 74 V, 2001
  • Bennink, M. L., Leuba, S. H., Leno, G. H., Zlatanova, J., de Grooth, B. G. and Greve, J. Unfolding individual nucleosomes by stretching single chromatin fibers with optical tweezers. Nature Struct. Biol. 8 606-610, 2001
  • Bennink, M. L, Pope, L. H., Leuba, S. H.,de Grooth, B. G. and Greve, J. Single chromatin fibre assembly using optical tweezers. Single Molecules 2 91-97, 2001
  • Karymov, M. A., Tomschik, M., Leuba, S. H., Caiafa, P. and Zlatanova, J. DNA Methylation-dependent chromatin fiber compaction in vivo and in vitro: Requirement for linker histone. FASEB J. 15 2631-2641, 2001
  • Tomschik, M., Karymov, M. A., Zlatanova, J., and Leuba, S. H. The archaeal histone-fold protein HMf organizes DNA into bona fide chromatin fibers. Struct. Fold. Des. 9 1201-1211, 2001
  • Leuba, S. H., Karymov, M. A., Liu, Y. Z., Lindsay, S. M. and Zlatanova, J. Mechanically stretching single chromatin fibers In Gene Therapy and Molecular Biology Boulikas, T., Ed. 4 297-301, 1999
  • Leuba, S. H. and Bustamante, C. Analysis of Chromatin by Scanning Force Microscopy. In Methods in Molecular Biology Becker, P., Ed 119 143-160, 1999
  • Liu, Y. Z., Leuba, S. H., and Lindsay, S. M. Relationship between stiffness and force in single molecule pulling experiments. Langmuir 15 8547-8548, 1999
  • An, W., Zlatanova, J., Leuba, S. H. and van Holde, K. The site of binding of linker histone to the nucleosome does not depend upon the amino termini of core histones. Biochemie 81 727-732, 1999
  • Zlatanova, J., Leuba, S. H. and van Holde, K. Chromatin structure revisited.Critical Reviews in Eukaryotic Gene Expression 9 245-255, 1999
  • Zlatanova, J., Yaneva, J. and Leuba, S. H. Proteins that specifically recognize cisplatin-damaged DNA: a clue to anticancer activity of cisplatin. FASEB J. 12(10) 791-799, 1998
  • van Holde, K., Leuba, S. H. and Zlatanova, J. Physical Approaches to the Study of Chromatin. In: Gene Therapy and Molecular Biology: From the Bench to the Cell Nucleus and to Clinical Applications, Boulikas, T., Ed.,1 475-482, 1998
  • Leuba, S. H., Bustamante, C., van Holde, K., and Zlatanova, J. Linker Histone tails and N-tails of histone H3 are redundant: scanning force microscopy studies of reconstituted fibers. Biophys. J. 74(6) 2830-2839, 1998b PDF file
  • Leuba, S. H., Bustamante, C., Zlatanova, J., and van Holde, K. Contributions of linker histones and histone H3 to chromatin structure: scanning force microscopy studies on trypsinized fibers. Biophys. J. 74(6) 2823-2829, 1998a PDF file
  • Zlatanova, J., Leuba, S. H., and van Holde, K. Chromatin fiber structure: morphology, molecular determinants, structural transitions. Biophys. J. 74(5) 2554-2566, 1998 PDF file
  • An, W., Leuba, S. H., van Holde, K., and Zlatanova, J. Linker histone protects DNA on only one side of the core particle and in a sequence-dependent manner. Proc. Natl. Acad. Sci. USA 95(7) 3396-3401, 1998
  • Leuba, S. H. and Zlatanova, J. Methods of Reconstitution of Linker Histone onto Oligonucleosomes. Methods in Molecular and Cellular Biology 6 116-124, 1997
  • Leuba, S. H. A One-Step Preparative Method to Reconstitute Linker Histones onto Linker Histone-Stripped Chromatin Fibers. Methods in Molecular and Cellular Biology 6 125-133, 1997
  • Yaneva, J., Leuba, S. H., van Holde, K. and Zlatanova, J. . The major chromatin protein histone H1 preferentially binds to cis-platinum damaged DNA. Proc. Natl. Acad. Sci. USA 94(25) 13448-13451, 1997
  • Bustamante, C., Zuccheri, G., Leuba, S. H., Yang, G. and Samori, B. Visualization and analysis of chromatin by scanning force microscopy. METHODS: A Companion to Methods in Enzymology 12(1) 73-83, 1997
  • Yang, G. Leuba, S. H. and Bustamante, C.Scanning Force Microscopy of Chromatin Fibers: A New Approach to an Old Problem. Biological Structure and Dynamics 1 155-172, 1995
  • Leuba, S. H., Yang, G., Robert, C., van Holde, K., Zlatanova, J. and C. Bustamante.Extended Chromatin Fibers: Evidence from Scanning Force Microscopy Studies. SPIE Proc. 2384 33-44, 1995
  • Zlatanova, J., Leuba, S. H., C. Bustamante, and van Holde, K. Role of the Structural Domains of the Linker Histones and Histone H3 in the Chromatin Fiber Structure at Low-ionic Strength: SFM Studies on Partially Trypsinized Chromatin. SPIE Proc. 2384 22-32, 1995
  • Yang, G., Leuba, S. H., Bustamante, C., van Holde, K., and Zlatanova, J. Scanning Force Microscopy Study of Native and Linker Histone Depleted Chromatin Fibers. SPIE Proc. 2384 13-21, 1995
  • Yang, G., Leuba, S. H., Bustamante, C., Zlatanova, J. and van Holde, K. Role of linker histones in extended chromatin fibre structure. Nat Struct Biol. 1(11) 761-763, 1994
  • Leuba, S. H., Yang, G., Robert, C., Samori, B., van Holde, K., Zlatanova, J. and C. Bustamante. Three-dimensional structure of extended chromatin fibers as revealed by tapping-mode scanning force microscopy. Proc. Natl. Acad. Sci. USA 91(24) 11621-11625, 1994
  • Zlatanova, J., Leuba, S. H., Yang, G., Bustamante, C. and K. van Holde. Linker DNA accessibility in chromatin fibers of different conformations: A reevaluation. Proc. Natl. Acad. Sci. USA 91(12) 5277-5280, 1994
  • Varga-Weisz, P., Zlatanova, J., Schroth, G. P., Leuba, S. H., and K. van Holde. Binding of histones H1 and H5 and their globular domains to four-way junction DNA. Proc. Natl. Acad. Sci. USA 91(9) 3525-3529, 1994
  • Leuba, S. H., Zlatanova, J. and K. van Holde. On the location of linker DNA in the chromatin fiber. Studies with immobilized micrococcal nuclease. J. Mol. Biol. 235(3) 871-880, 1994
  • Krylov, D., Leuba, S., van Holde, K. and J. Zlatanova. Histones H1 and H5 interact+ preferentially with cross-overs of double-helical DNA. Proc. Natl. Acad. Sci. USA 90(11) 5052-5056, 1993
  • Leuba, S. H., Zlatanova, J. and K. van Holde. On the Location of histones H1 and H5 in the chromatin fiber. Studies with immobilized trypsin and chymotrypsin. J. Mol. Biol. 229 917-929, 1993
  • Garcia-Ramirez, M., Leuba, S. H. and Ausio, J. One-step fractionation method for isolating H1 histones from chromatin under nondenaturing conditions. Protein Expr Purif. 1(1) 40-44, 1990

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  Dr. Sanford H. Leuba
  Laboratory of Receptor Biology and Gene Expression
  NIH NCI
  41 Library Dr MSC 5055 RM B602
  BETHESDA, MD 20892-5055
  (301) 496-7528 Voice
  (301) 496-4951 FAX
  leuba@nih.gov

  Last revised on March 13, 2001
  by Dawn Walker

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