Alkaline gel electrophoresis of deoxyribonucleic acid photoreacted with trimethylpsoralen: rapid and sensitive detection of interstrand cross-links.
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.
D010582:Bacteriophage lambda; D010584:Bacteriophage phi X 174; D055598:Chemical Phenomena; D002621:Chemistry; D004262:DNA Restriction Enzymes; D004279:DNA, Viral / Q000528:radiation effects*; D015247:Deoxyribonuclease HindIII; D015252:Deoxyribonucleases, Type II Site-Specific; D004587:Electrophoresis, Agar Gel; D011564:Furocoumarins; D006863:Hydrogen-Ion Concentration; D008027:Light; D008854:Microscopy, Electron; D009691:Nucleic Acid Denaturation; D014307:Trioxsalen
D004279:DNA, Viral; D011564:Furocoumarins; D004262:DNA Restriction Enzymes; D015247:Deoxyribonuclease HindIII; D015252:Deoxyribonucleases, Type II Site-Specific; C024403:GGCC-specific type II deoxyribonucleases; D014307:Trioxsalen