Crosslinking of proteins to DNA in human nuclei using a 60 femtosecond 266 nm laser

• Published in: Nucleic acids research Vol. 27; no. 18; pp. 3676 - 3684
• Main Authors: Lejnine, S., Durfee, G., Murnane, M., Kapteyn, H. C., Makarov, V. L., Langmore, J. P.
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 15-09-1999; Oxford Publishing Limited (England)
• Subjects: Antigens, Nuclear; Base Sequence; Cell Line; Cell Nucleus - genetics; Cell Nucleus - metabolism; Cell Nucleus - radiation effects; Centrifugation, Density Gradient; DNA - analysis; DNA - genetics; DNA - metabolism; DNA Damage - genetics; DNA Damage - radiation effects; DNA Helicases; DNA Restriction Enzymes - metabolism; DNA, Ribosomal - analysis; DNA, Ribosomal - genetics; DNA, Ribosomal - metabolism; DNA-Binding Proteins - analysis; DNA-Binding Proteins - metabolism; Dose-Response Relationship, Radiation; Histones - analysis; Histones - metabolism; Humans; Kinetics; Ku Autoantigen; Lasers; Micrococcal Nuclease - metabolism; Nuclear Proteins - analysis; Nuclear Proteins - metabolism; Precipitin Tests; Protein Binding; Telomere - genetics; Telomere - metabolism
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/27.18.3676

We developed appropriate conditions to use a laser with 60 femtosecond pulses, a frequency of 1 KHz and a wavelength of 266 nm to efficiently crosslink proteins to DNA in human nuclei for the purpose of using immunoprecipitation to study the binding of specific proteins to specific sequences of DNA under native conditions. Irradiation of nuclei for 30 min with 1–3 GW/cm2 pulses crosslinked 10–12% of total protein to DNA. The efficiency of crosslinking was dose and protein specific. Histones H1 and H3 were crosslinked by 15 min of irradiation with 20–25% efficiency, at least 10 times more strongly than the other histones, consistent with experiments using conventional UV light. Irradiation for 15 min did not damage proteins, as assayed by SDS-PAGE of Ku-70 and histones. Although the same level of irradiation did not cause double-strand breaks, it did make the DNA partially insensitive to EcoRI restriction enzyme, probably through formation of thymidine dimers. Immuno-analysis of crosslinked nucleoprotein showed that Ku crosslinking to nuclear DNA is detectable only in the presence of breaks in the DNA, and that nucleosomes are bound to a significant fraction of the telomeric repeat (TTAGGG)n.