Using optical tweezers to study protein-DNA interactions

Mechanical manipulation of single DNA molecules can provide novel information about protein-DNA interactions. Here we review two examples studied by our group. First, we have studied the forced unraveling of nucleosomes assembled on heterogeneous DNA using core histones, the histone chaperone NAP-1, and ATP-dependent chromatin assembly and remodeling factor (ACF). We measure abrupt events releasing ~55 to 95 base pairs of DNA, which are attributable to non-equilibrium unraveling of individual nucleosomes. Wide variations observed in the unraveling force and sudden DNA re-wrapping events may have an important regulatory influence on DNA directed biochemical processes. Second, we have studied the mechanics and dynamics of single DNA looping and cleavage by "two-site" restriction enzymes. Cleavage is measured as a function of DNA tension, incubation time, and enzyme concentration, distinguishing enzymes that require DNA looping from ones that do not. Forced disruption of fixed DNA loops formed in the absence of Mg2+ is observed, allowing the distribution of number of loops, loop length, and disruption force to be measured as a function of time, DNA tension, and ionic conditions.

[1]  F. Thoma,et al.  Chromatin reconstituted from tandemly repeated cloned DNA fragments and core histones: A model system for study of higher order structure , 1985, Cell.

[2]  J. T. Kadonaga,et al.  Chromatin Assembly by Dna-translocating Motors , 2022 .

[3]  Vasily M. Studitsky,et al.  Overcoming a nucleosomal barrier to transcription , 1995, Cell.

[4]  J. Perona,et al.  Role of protein-induced bending in the specificity of DNA recognition: crystal structure of EcoRV endonuclease complexed with d(AAAGAT) + d(ATCTT). , 1998, Journal of molecular biology.

[5]  H. Schiessel,et al.  DNA spools under tension. , 2003, Physical review letters.

[6]  S. Kowalczykowski,et al.  Unwinding of Nucleosomal DNA by a DNA Helicase (*) , 1995, The Journal of Biological Chemistry.

[7]  S. Smith,et al.  Single-molecule studies of DNA mechanics. , 2000, Current opinion in structural biology.

[8]  D. Hogness,et al.  Spatial and temporal patterns of E74 transcription during Drosophila development , 1990, Cell.

[9]  James T Kadonaga,et al.  Chromatin remodeling by ATP-dependent molecular machines. , 2003, BioEssays : news and reviews in molecular, cellular and developmental biology.

[10]  Michelle D. Wang,et al.  Specific contributions of histone tails and their acetylation to the mechanical stability of nucleosomes. , 2005, Journal of molecular biology.

[11]  Jan Greve,et al.  Unfolding individual nucleosomes by stretching single chromatin fibers with optical tweezers , 2001, Nature Structural Biology.

[12]  S. Halford,et al.  Enzyme-mediated DNA looping. , 2004, Annual review of biophysics and biomolecular structure.

[13]  T. Richmond,et al.  Crystal structure of the nucleosome core particle at 2.8 Å resolution , 1997, Nature.

[14]  Jonathan Widom,et al.  Spontaneous sharp bending of double-stranded DNA. , 2004, Molecular cell.

[15]  D M Crothers,et al.  Artificial nucleosome positioning sequences. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[16]  A. Pingoud,et al.  Sau3AI, a Monomeric Type II Restriction Endonuclease That Dimerizes on the DNA and Thereby Induces DNA Loops* , 2001, The Journal of Biological Chemistry.

[17]  Claudio Nicolini,et al.  Chromatin Structure and Function , 1979, NATO Advanced Study Institutes Series.

[18]  C. Bustamante,et al.  Pulling a single chromatin fiber reveals the forces that maintain its higher-order structure. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[19]  Michelle D. Wang,et al.  Mechanical disruption of individual nucleosomes reveals a reversible multistage release of DNA , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[20]  C. Bustamante,et al.  An integrated laser trap/flow control video microscope for the study of single biomolecules. , 2000, Biophysical journal.

[21]  R Schleif.  DNA looping. , 1992, Annual review of biochemistry.

[22]  J. Widom,et al.  Role of DNA sequence in nucleosome stability and dynamics , 2001, Quarterly Reviews of Biophysics.

[23]  A. Wolffe,et al.  Regulation of Chromatin Structure and Function , 1994 .

[24]  J. T. Kadonaga,et al.  Strategies for the reconstitution of chromatin , 2004, Nature Methods.

[25]  E. Siggia,et al.  Driving proteins off DNA using applied tension. , 1997, Biophysical journal.

[26]  J. Widom,et al.  Effects of histone acetylation on the equilibrium accessibility of nucleosomal DNA target sites. , 2001, Journal of molecular biology.

[27]  James T Kadonaga,et al.  Chromatin assembly in vitro with purified recombinant ACF and NAP-1. , 2003, Methods in enzymology.

[28]  Sarah C. R. Elgin,et al.  Chromatin structure and gene expression , 1995 .

[29]  A. Aggarwal,et al.  Structure of Bam HI endonuclease bound to DNA: partial folding and unfolding on DNA binding. , 1995, Science.

[30]  C. Bustamante,et al.  Overstretching B-DNA: The Elastic Response of Individual Double-Stranded and Single-Stranded DNA Molecules , 1996, Science.

[31]  Ryuji Kobayashi,et al.  ACF, an ISWI-Containing and ATP-Utilizing Chromatin Assembly and Remodeling Factor , 1997, Cell.