title: Molecular Dissection of Chromatin Maturation via Click Chemistry.
abstract: DNA synthesis and chromatin assembly are the two most critical processes of eukaryotic cell division. It is well known that their coordination is tightly regulated. Although the interplay between DNA and its higher-order chromatin state is integral for many processes, including cell survival and genome stability, little is known about the re-establishment of chromatin structure during the cell cycle. Moreover, the extent to which the fidelity of the newly synthesized chromatin plays a role in the maintenance of cellular identity is still under debate. Here, we present a novel approach to purify nascent chromatin from the replication fork. In this protocol, we take advantage of click chemistry, a method that allows efficient conjugation of azide-containing biotin molecules to ethynyl-labeled nucleic acids. Using this approach, we selectively enrich biotin-nucleic acid conjugates via streptavidin affinity purification to pull down and assess chromatin states as well as chromatin-bound complexes from newly replicated DNA fragments.
authors.affiliation: Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts.|Department of Genetics, Harvard Medical School, Boston, Massachusetts.
authors.forename: Ozlem
authors.initials: O
authors.lastname: Yildirim
authors.affiliation: Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts.|Department of Genetics, Harvard Medical School, Boston, Massachusetts.
authors.forename: Robert E
authors.initials: RE
authors.lastname: Kingston
mesh_terms: D002843:Chromatin / Q000737:chemistry* / Q000302:isolation & purification*; D057930:Click Chemistry / Q000379:methods; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods
chemical_list: D002843:Chromatin; D004247:DNA
keywords: EdU; cell cycle; click chemistry; epigenetic inheritance; nascent chromatin