Double-stranded cleavage of pBR322 by a diiron complex via a 'hydrolytic' mechanism

L. M.T. Schnaith, R. S. Hanson, L. Que

Research output: Contribution to journalArticlepeer-review

107 Scopus citations

Abstract

Treatment of plasmid pBR322 with Fe2-(HPTB)(OH)(NO3)4(HPTB = N,N,N',N'- tetrakis(2-benzimidazolylmethyl)-2-hydroxy-1,3-diaminopropane) and H2O2 or O2 and a reductant (dithiothreitol or ascorbate) results in double-stranded cleavage of the plasmid. The linearization of supercoiled pBR322 by this complex is not inhibited by hydroxyl radical scavengers. On the other hand, the linearized pBR322 is efficiently religated by T4 DNA ligase, and the presence of 3'-OH and 5'-OPO3 ends is corroborated by 3'- and 5'-end- labeling studies. These observations indicate that cleavage results from hydrolysis of the DNA-phosphate backbone, which is proposed to occur by nucleophilic attack of the bound peroxide on the phosphodiester. Double- stranded cleavage by the Fe2(HPTB)(OH)(NO3)4/H2O2 adduct preferentially occurs between bp 3489 and 3485 of pBR322.

Original languageEnglish (US)
Pages (from-to)569-573
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume91
Issue number2
DOIs
StatePublished - 1994

Keywords

  • DNA-cleaving agent
  • hydrolysis

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