Crosslinking of proteins to DNA in human nuclei using a 60 femtosecond 266 nm laser.
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/cm(2)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 Eco RI 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).
Lejnine
S
S
Biophysics Research Division, University of Michigan, Ann Arbor 48109-1055, USA.
Durfee
G
G
Murnane
M
M
Kapteyn
H C
HC
Makarov
V L
VL
Langmore
J P
JP
D034961:Antigens, Nuclear; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D002499:Centrifugation, Density Gradient; D004247:DNA / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage / Q000235:genetics / Q000528:radiation effects; D004265:DNA Helicases; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004275:DNA, Ribosomal / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000032:analysis / Q000378:metabolism*; D004307:Dose-Response Relationship, Radiation; D006657:Histones / Q000032:analysis / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D000072200:Ku Autoantigen; D007834:Lasers; D008836:Micrococcal Nuclease / Q000378:metabolism; D009687:Nuclear Proteins / Q000032:analysis / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D016615:Telomere / Q000235:genetics / Q000378:metabolism
D034961:Antigens, Nuclear; D004275:DNA, Ribosomal; D004268:DNA-Binding Proteins; D006657:Histones; D009687:Nuclear Proteins; D004247:DNA; D004262:DNA Restriction Enzymes; D008836:Micrococcal Nuclease; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen