Solomon, Mark J. and Varshavsky, Alexander (1985) Formaldehyde-mediated DNA-protein crosslinking: A probe for in vivo chromatin structures. Proceedings of the National Academy of Sciences of the United States of America, 82 (19). pp. 6470-6474. ISSN 0027-8424. http://resolver.caltech.edu/CaltechAUTHORS:SOLpnas85
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Formaldehyde (HCHO) produces DNA-protein crosslinks both in vitro and in vivo. Simian virus 40 (SV40) chromosomes that have been fixed by prolonged incubation with HCHO either in vitro or in vivo (within SV40-infected cells) can be converted to nearly protein-free DNA by limit-digestion with Pronase in the presence of NaDodSO4. The remaining Pronase-resistant DNA-peptide adducts retard the DNA upon gel electrophoresis, allowing resolution of free and crosslink-containing DNA. Though efficiently crosslinking histones to DNA within nucleosomes both in vitro and in vivo, HCHO does not crosslink either purified lac repressor to lac operator-containing DNA or an (A + T)-DNA-binding protein ( -protein) to its cognate DNA in vitro. Furthermore, a protein that does not bind to DNA, such as serum albumin, is not crosslinked to DNA by HCHO even at extremely high protein concentrations. These properties of HCHO as a DNA-protein crosslinker are used to probe the distribution of nucleosomes in vivo. We show that there are no HCHO-crosslinkable DNA-protein contacts in a subset of SV40 chromosomes in vivo within a 325-base-pair stretch that spans the "exposed" (nuclease-hypersensitive) region of the SV40 chromosome. This replication origin-proximal region has been found previously to lack nucleosomes in a subset of isolated SV40 chromosomes. We discuss other applications of the HCHO technique, including the possibility of obtaining base-resolution in vivo nucleosome "footprints."
|Additional Information:||Copyright © 1985 by the National Academy of Sciences. Communicated by Gary Felsenfeld, June 6, 1985. We are greatly indebted to Kathleen Matthews, Frangois Strauss, and James Wang for their gifts of lac repressor, alpha-protein, and plasmid pJW270, respectively. We also thank Daniel Finley for helpful comments on the manuscript and Barbara Doran for secretarial assistance. This work was supported by a grant to A.V. from the National Cancer Institute (CA30367). M.S. was supported by a predoctoral fellowship from the National Science Foundation. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.|
|Subject Keywords:||simian virus 40 chromosomes; nucleosome-free region; Pronase-resistant crosslinks; lac repressor; alpha-protein|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Tony Diaz|
|Deposited On:||30 Jan 2006|
|Last Modified:||26 Dec 2012 08:45|
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