Measurement of interstrand cross-link frequency and distance between interruptions in DNA exposed to 4,5',8-trimethylpsoralen and near-ultraviolet light.
Bifunctional psoralens react photochemically with DNA to form single-strand adducts and interstrand, chemical cross-links. Cross-link formation is first order with [P], the concentration of added psoralen, when [P] much less than Kd, the psoralen-DNA dissociation constant. DNA molecules containing interstrand cross-links are reversibly bihelical and so are readily detected. It was not heretofore possible to determine cross-link frequency in polydisperse DNA from the mass F of DNA spared cross-linkage. We have derived a statistical relation to calculate cross-link frequency at fixed light exposure and variable [P]. We show here that S, the initial slope of the curve described by -ln F as a function of [P], is proportional to Mw, the weight-average molecular weight of nick-free DNA. The cross-link frequency at any [P] can be determined from k, a constant measured for DNA of known Mw at low cross-linkage. This relation is valid for DNA of any molecular weight distribution. In experiments with uniform length DNA, -ln F (cross-link frequency) increased in simple proportion to [P]. Intact and restriction endonuclease HindIII digested phage lambda DNA molecules have discrete lengths. S for each was proportional to Mw of the twin helix even though the molecular weight distribution of the restriction fragments was skewed. S was proportional to Mw and to the median molecular weight of sheared cellular DNA over a wide range. Also, we found that 1/S was linear with exposure of cellular DNA to gamma radiation. S can therefore be used to calculate L, the average distance between interruptions in the double helix.
Matsuo
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Ross
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D000818:Animals; D002460:Cell Line; D004247:DNA / Q000187:drug effects* / Q000528:radiation effects; D004926:Escherichia coli; D011564:Furocoumarins / Q000494:pharmacology*; D007700:Kinetics; D008433:Mathematics; D008970:Molecular Weight; D010957:Plasmids / Q000187:drug effects* / Q000528:radiation effects; D014307:Trioxsalen / Q000494:pharmacology*; D014466:Ultraviolet Rays
D011564:Furocoumarins; D004247:DNA; D014307:Trioxsalen