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

Abstract

Formaldehyde (HCHO) produces DNAprotein 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 viio, HCHO does not crosslink either purified lac repressor to lac operator-containing DNA or an (A + T)-DNA-binding protein (a-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."DNA-protein interactions can be probed in vitro by a number of techniques, including chemical modification (1) and nuclease-or drug-mediated "footprinting" (2-5). Most of these assays are kinetic in nature, since the probes used are not absolutely specific for the structure of interest. In a different set of approaches, DNA-bound proteins are first crosslinked to DNA by treatments with UV light (6), formaldehyde (HCHO) (7), dimethyl sulfate (8), or a variety of other agents (9). Some of the above methods, in particular DNA modification by dimethyl sulfate (10,11) On the other hand, HCHO produces DNA-protein crosslinks both in vitro and in vivo (7,(13)(14)(15)(16)(17)(18) and at the same time displays virtually no reactivity toward free double-stranded DNA (19,20). Since HCHO produces DNA-protein (7,(13)(14)(15)(16), , and protein-protein (18) crosslinks, its addition to living cells results within minutes in formation of crosslinked networks of biopolymers, thus preventing any large-scale redistribution of cellular components upon prolonged ("limit") fixation. HCHO-induced crosslinks, in particular DNA-protein crosslinks, can be reversed under relatively mild conditions, as demonstrated previously (16,18) and further refined in the present work.Limit-digestion of HCHO-fixed eukaryotic chromosomes with relatively nonspecific proteinases, such as Pronase or proteinase K, does not yield a completely peptide-free DNA (7,14,21). Moreover, DNA fragments containing Pronaseresistant peptide-DNA adducts have reduced electrophoretic mobilities (7). We hav...

Specific RNP capture with antisense LNA/DNA mixmers

“…However, it may be important to conduct independent analyses of the individual probes to ascertain similar specificity and stringency as with the single probe approach. To increase the yield of the protein components of the RNPs, this protocol could be combined with more highly efficient crosslinkers such as formaldehyde (Solomon and Varshavsky 1985;Niranjanakumari et al 2002).…”

Characterization of the mycobacterial chromosome segregation protein ParB and identification of its target in Mycobacterium smegmatis

“…M. smegmatis and its parB deletion mutant cultivated for 48 h in modified Sauton's broth (Mordarska et al, 1972) were cross-linked by addition of formaldehyde (final concentration: 1 %) for 5 min as described previously (Solomon & Varshavsky, 1985). Cells not subjected to cross-linking and mutant strains served as negative controls and were treated subsequently in the same way as the experimental samples.…”

Formation, Accumulation, and Hydrolysis of Endogenous and Exogenous Formaldehyde-Induced DNA Damage

“…All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com bind and induce DNA monoadducts (McGhee and von Hippel, 1977), DNA-DNA crosslinks (Lu et al, 2010a), DNA-protein crosslinks (DPCs) (Casanova-Schmitz and Heck, 1983;MaganaSchwencke and Ekert, 1978;Solomon and Varshavsky, 1985), and DNA-glutathione crosslinks . The carcinogenic mode of action is based on the synergism among the formation of DNA mono-adducts, DPCs, and increased cell proliferation that lead to mutations (International Agency for Research on Cancer, 2006).…”