UV light-induced crosslinking of short DNA duplex strands: nucleotide sequence preferences and a prominent role of the duplex ends.
By various doses of UV light, we irradiated 32 short DNA duplexes having between 12 and 40 nucleotide residues in length, and monitored the induced crosslinks between the complementary DNA strands by denaturing polyacrylamide gel electrophoresis. The experiments revealed that the crosslinking was strongest with the alternating sequence of T and A and weaker with the alternating sequence of T and G (C and A in the complementary strand). On the other hand, GC blocks of any sequence provided undetectable amounts of interstrand crosslinks even at the highest doses of UV irradiation. The amount of crosslinked strands logarithmically increased with the UV dose but it did not depend on the oligonucleotide concentration, ionic strength, divalent magnesium or manganese cations and pH at least within the examined regions of the experimental conditions, unless the oligonucleotide denatured or isomerized into a unimolecular foldback. The extent of crosslinking also did not depend on the (dT-dA)n duplex length to indicate that the crosslink was predominantly localized at a specific duplex locus. Experiments with (dT-dA)8 "mutants" in which AT pairs were systematically replaced by GC pairs at various molecule positions, revealed that the crosslinking predominantly occurred at the oligo(dT-dA) duplex ends. The crosslinking is a direct method to detect duplexes of DNA, which is here, for example, demonstrated with the heteroduplex of (dT-dA)12 and (dT-dA)16.
Kovanda
J
J
Institute of Biophysics, Czech Academy of Sciences, Brno, Czech Republic.
Kejnovský
E
E
Arnold
L
L
Kypr
J
J
D004591:Electrophoresis, Polyacrylamide Gel; D009690:Nucleic Acid Conformation / Q000528:radiation effects; D009691:Nucleic Acid Denaturation; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry* / Q000528:radiation effects*; D016385:TATA Box; D013696:Temperature; D014466:Ultraviolet Rays
D009692:Nucleic Acid Heteroduplexes