Accurate measurement of psoralen-crosslinked DNA: direct biochemical measurements and indirect measurement by hybridization.
This paper evaluates methods to measure crosslinkage due to psoralen plus light in total DNA and in specific sequences. DNA exposed in cells or in vitro to a bifunctional psoralen and near ultraviolet light accumulates interstrand crosslinks. Crosslinkage is the DNA mass fraction that is attached in both strands to a crosslink. We show here biochemical methods to measure psoralen photocrosslinkage accurately in total DNA. We also describe methods to measure photocrosslinkage indirectly, in specific sequences, by nucleic acid hybridization. We show that a single 4,5',8-trimethylpsoralen (TMP) crosslink causes at least 50 kbp of alkali-denatured DNA contiguous in both strands with it to snap back into the duplex form when the denatured preparation is returned to neutral pH. This process was so efficient that the DNA was not nicked by the single-strand nuclease S1 at 100-fold excess after snapping back. Uncrosslinked DNA was digested to acid-soluble material by the enzyme. Crosslinkage therefore equals the fraction of S1-resistant nucleotide in this kind of experiment. We alkali-denatured DNA samples crosslinked to varying degrees by varying TMP concentration at constant light exposure. We then measured crosslinkage by ethidium bromide (EtBr) fluorometry at pH 11.8; by EtBr fluorometry at neutral pH of S1 digests of the DNA; and by the fraction of radioactivity remaining acid insoluble in S1-digests of DNA labeled uniformly with [3H]deoxythymidine. These assays measure distinct physical properties of crosslinked DNA. Numerical agreement is expected only when all three measurements are accurate. Under optimum conditions, the three methods yielded identical results over the range of measurement. Using alkaline EtBr fluorescence in crude cell lysates, we detected crosslinks at frequencies in the range of 1.6 X 10(-7) per base pair. These levels were compatible with cell survival, attesting to the sensitivity of the measurement system. Crosslinkage affected hybridization as well. One crosslink prevented all alkali-denatured DNA contiguous in both strands with it from hybridizing to complementary DNA either on solid supports or in solution. Strand-length effects on crosslinkage and on reassociation caused solution hybridization levels to exceed those predicted by simple theory. In a quantitative, dot-blotting assay hybridization was linear up to membrane saturation by denatured, uncrosslinked DNA of any strand length.(ABSTRACT TRUNCATED AT 400 WORDS)
D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D004247:DNA / Q000032:analysis* / Q000528:radiation effects; D004269:DNA, Bacterial / Q000032:analysis; D004277:DNA, Single-Stranded / Q000032:analysis; D004331:Drosophila melanogaster / Q000032:analysis; D004926:Escherichia coli / Q000032:analysis; D005363:Ficusin / Q000494:pharmacology*; D011564:Furocoumarins / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D010777:Photochemistry; D010957:Plasmids
D003432:Cross-Linking Reagents; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D011564:Furocoumarins; D004247:DNA; D005363:Ficusin