DOUBLE-STRANDED CLEAVAGE OF PBR322 BY A DIIRON COMPLEX VIA A HYDROLYTIC MECHANISM

Treatment of plasmid pBR322 with Fe-2-(HPTB)(OH)(NO3)4(HPTB=N,N,N',N'-tetrakis(2-benzimidazolylmethyl) 2-hydroxy-1,3 -diaminopropane) and H2O2 or O-2 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 Fe-2(HPTB)(OR)(NO3)(4)/H2O2 adduct preferentially occurs between bp 3489 and 3485 of pBR322.