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.
- Research Organization:
- Rockefeller Univ., New York, NY (USA)
- OSTI ID:
- 6583894
- Journal Information:
- Arch. Biochem. Biophys.; (United States), Vol. 266:2
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
CROSS-LINKING
MEASURING METHODS
DNA
PSORALEN
GENETIC EFFECTS
ESCHERICHIA COLI
FLUORESCENCE
GENETIC RADIATION EFFECTS
HYBRIDIZATION
IN VITRO
IN VIVO
NUCLEIC ACID DENATURATION
PLASMIDS
THYMIDINE
TRACER TECHNIQUES
TRITIUM COMPOUNDS
ULTRAVIOLET RADIATION
ANTICOAGULANTS
AZINES
BACTERIA
BIOLOGICAL EFFECTS
BIOLOGICAL RADIATION EFFECTS
CELL CONSTITUENTS
CHEMICAL REACTIONS
COUMARINS
DRUGS
ELECTROMAGNETIC RADIATION
HEMATOLOGIC AGENTS
HETEROCYCLIC COMPOUNDS
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LUMINESCENCE
MICROORGANISMS
NUCLEIC ACIDS
NUCLEOSIDES
NUCLEOTIDES
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC OXYGEN COMPOUNDS
POLYMERIZATION
PYRIMIDINES
RADIATION EFFECTS
RADIATIONS
RIBOSIDES
550201* - Biochemistry- Tracer Techniques
560120 - Radiation Effects on Biochemicals
Cells
& Tissue Culture