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Sci Rep. 2017 Sep 15;7(1):11725. doi: 10.1038/s41598-017-12010-5.

Time-resolved analysis of DNA-protein interactions in living cells by UV laser pulses.

Author information

1
Dipartimento di Biochimica, Biofisica e Patologia Generale, Università degli Studi della Campania "L. Vanvitelli", Vico L. De Crecchio 7, 80138, Napoli, IT, Italy. angela.nebbioso@unicampania.it.
2
Dipartimento di Biochimica, Biofisica e Patologia Generale, Università degli Studi della Campania "L. Vanvitelli", Vico L. De Crecchio 7, 80138, Napoli, IT, Italy.
3
IRCCS SDN, Via E. Gianturco, 113, 80143, Napoli, IT, Italy.
4
Department of Molecular Biology, NCMLS, Radboud University, 6500, Nijmegen, NL, Netherlands.
5
Dipartimento di Fisica, Università di Napoli Federico II, Via Cinthia, 80100, Napoli, IT, Italy.
6
Dipartimento di Fisica, Università di Napoli Federico II, Via Cinthia, 80100, Napoli, IT, Italy. carlo.altucci@unina.it.
7
Dipartimento di Biochimica, Biofisica e Patologia Generale, Università degli Studi della Campania "L. Vanvitelli", Vico L. De Crecchio 7, 80138, Napoli, IT, Italy. lucia.altucci@unicampania.it.

Abstract

Interactions between DNA and proteins are mainly studied through chemical procedures involving bi-functional reagents, mostly formaldehyde. Chromatin immunoprecipitation is used to identify the binding between transcription factors (TFs) and chromatin, and to evaluate the occurrence and impact of histone/DNA modifications. The current bottleneck in probing DNA-protein interactions using these approaches is caused by the fact that chemical crosslinkers do not discriminate direct and indirect bindings or short-lived chromatin occupancy. Here, we describe a novel application of UV laser-induced (L-) crosslinking and demonstrate that a combination of chemical and L-crosslinking is able to distinguish between direct and indirect DNA-protein interactions in a small number of living cells. The spatial and temporal dynamics of TF bindings to chromatin and their role in gene expression regulation may thus be assessed. The combination of chemical and L-crosslinking offers an exciting and unprecedented tool for biomedical applications.

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