Formaldehyde-mediated DNA-protein crosslinking: a probe for in vivo chromatin structures

Journal Article · Tue Oct 01 00:00:00 EDT 1985 · Proc. Natl. Acad. Sci. U.S.A.; (United States)

DOI:https://doi.org/10.1073/pnas.82.19.6470· OSTI ID:6385619

Solomon, M J; Varshavsky, A

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 NaDodSO/sub 4/. 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 (..cap alpha..-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. It is shown 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 chromosomes. This replication origin-proximal region has been found previously to lack nucleosomes in a subset of isolated SV40 chromosomes. Other applications of the HCHO technique are discussed, including the possibility of obtaining base-resolution in vivo nucleosome footprints.

• Research Organization: Massachusetts Institute of Technology, Cambridge
• OSTI ID: 6385619
• Journal Information: Proc. Natl. Acad. Sci. U.S.A.; (United States), Vol. 82:19
• Country of Publication: United States
• Language: English

Related Subjects

63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
59 BASIC BIOLOGICAL SCIENCES
DNA
CROSS-LINKING
FORMALDEHYDE
GENETIC EFFECTS
CHROMATIN
CHROMOSOMES
ELECTROPHORESIS
IN VIVO
PEPTIDE HYDROLASES
PROTEIN STRUCTURE
SIMIAN VIRUS
ALDEHYDES
BIOLOGICAL EFFECTS
CHEMICAL REACTIONS
ENZYMES
HYDROLASES
MICROORGANISMS
NUCLEIC ACIDS
ORGANIC COMPOUNDS
PARASITES
POLYMERIZATION
VIRUSES
560301* - Chemicals Metabolism & Toxicology- Cells- (-1987)
550400 - Genetics