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

Treatment of plasmid pBR322 with )4(HPTB=N,N,N',N'-tetrakis(2-benzimid azolylmethyl) 2-hydroxy-1,3 -diaminopropane) and H2O2 or O-2 and a red uctant (dithiothreitol or ascorbate) results in double-stranded cleava ge of the plasmid. The linearization of supercoiled pBR322 by this com plex is not inhibited by hydroxyl radical scavengers. On the other han d, the linearized pBR322 is efficiently religated by T4 DNA ligase, an d the presence of 3'-OH and 5'-OPO3 ends is corroborated by 3'- and 5' -end-labeling studies. These observations indicate that cleavage resul ts from hydrolysis of the DNA-phosphate backbone, which is proposed to occur by nucleophilic attack of the bound peroxide on the phosphodies ter. Double-stranded cleavage by the Fe-2(HPTB)(OR)(NO3)(4)/H2O2 adduc t preferentially occurs between bp 3489 and 3485 of pBR322.