Alkaline gel electrophoresis of deoxyribonucleic acid photoreacted with trimethylpsoralen: rapid and sensitive detection of interstrand cross-links

Biochemistry. 1981 Mar 17;20(6):1431-7. doi: 10.1021/bi00509a005.

Abstract

Restriction fragments of phage lambda and phi X174 deoxyribonucleic acid (DNA) were photoreacted with 4,5',8-trimethylpsoralen to various extents, and the amount of covalent cross-linking was determined by electron microscopy of the DNA under totally denaturing conditions. The DNA was then analyzed by electrophoresis in alkaline agarose gels. A single cross-link in a DNA molecule produced a large decrease in its electrophoretic mobility. With DNA fragments 0.3--4 kilobase pairs in size, the apparent Mr (molecular weight) of the cross-linked DNA was 2.0 +/- 0.1 times and Mr of the unreacted, single-stranded DNA. A single cross-link in a larger DNA molecule resulted in an even greater increase in apparent Mr. Further cross-linking produced a decrease in the apparent Mr of the DNA, reaching a plateau at a value of 1.4 +/- 0.1 times the Mr of the unreacted, single-stranded DNA over a large range of fragment sizes (0.6--10 kilobase pairs). The apparent Mr of the cross-linked DNA was weakly dependent on the percentage of agarose in the gel. Although highly sensitive to interstrand cross-links the electrophoretic mobilities appeared to be unaffected by low levels of monoadducts (trimethylpsoralen covalently bound to one strand of the DNA). The DNA bandwidths increased by as much as 4-fold at low extents of cross-linking, presumably due to heterogeneity in the locations of the cross-links in the DNA molecules. The bands became sharp again at high levels of reaction. These observations from the basis of a new assay for interstrand DNA cross-links that is both more sensitive and more convenient than previous methods.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Bacteriophage lambda*
  • Bacteriophage phi X 174*
  • Chemical Phenomena
  • Chemistry
  • DNA Restriction Enzymes
  • DNA, Viral / radiation effects*
  • Deoxyribonuclease HindIII
  • Deoxyribonucleases, Type II Site-Specific*
  • Electrophoresis, Agar Gel
  • Furocoumarins*
  • Hydrogen-Ion Concentration
  • Light
  • Microscopy, Electron
  • Nucleic Acid Denaturation
  • Trioxsalen*

Substances

  • DNA, Viral
  • Furocoumarins
  • DNA Restriction Enzymes
  • Deoxyribonuclease HindIII
  • Deoxyribonucleases, Type II Site-Specific
  • GGCC-specific type II deoxyribonucleases
  • Trioxsalen