Large inverted duplications in amplified DNA of mammalian cells form hairpins in vitro upon DNA extraction but not in vivo.
I have analysed the duplex-to-hairpin transition of large inverted duplications with a short asymmetric center which are found in the amplified DNA of two mammalian cell lines resistant to cytotoxic drugs. Psoralen crosslinking experiments establish that this transition does not occur in vivo, but takes place in a significant portion of the palindromes during genomic DNA purification, at the phenol-chloroform extraction step. The introduction of single strand nicks in the DNA by gamma irradiation prior to its purification does not prevent hairpin formation but instead facilitates it. These results show that the rate-limiting step of the duplex-to-hairpin transition does not require negative supercoiling, and that transient melting of large segments of cellular DNA occurs during phenol-chloroform extraction. I also show, and discuss the fact, that only cellular DNA, and not cloned palindromic DNA, is able to undergo hairpin formation by this mechanism. These results bear practical implications for the study of inverted repeated DNA sequences in eukaryotic cells.
Hyrien
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Unité de Génétique Somatique, UA CNRS, Institut Pasteur, Paris, France.
D000659:AMP Deaminase / Q000235:genetics; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D003432:Cross-Linking Reagents; D004247:DNA / Q000187:drug effects / Q000235:genetics* / Q000528:radiation effects; D015342:DNA Probes; D005720:Gamma Rays; D005784:Gene Amplification; D005796:Genes; D007700:Kinetics; D005810:Multigene Family; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D014307:Trioxsalen / Q000494:pharmacology; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D015342:DNA Probes; D004247:DNA; D000659:AMP Deaminase; D014307:Trioxsalen