MECHANISM OF SITE-SELECTIVE DNA NICKING BY THE HYDRODIOXYL (PERHYDROXYL) RADICAL

In previous studies, the ability of the hydrodioxyl (perhydroxyl) radi cal [HOO., the conjugate acid of superoxide (O-2(.-))] to ''nick'' DNA under biomimetic conditions was demonstrated, and a sequence selectiv ity was observed. A background level of nonspecific nicking also was n oted. This paper provides support for 5'-hydrogen atom abstraction fro m the deoxyribose ring as the initial event in the sequence-selective nicking by O-2(.-)/HOO.. Two experiments support the proposed mechanis m. First, using a defined sequence 5'-P-32-labeled restriction fragmen t as the DNA substrate, only free (unalkylated) 3'-phosphate is produc ed at the site of nicking, Second, using poly (dA). poly (T) as the su bstrate, furfural is formed in the reaction from deoxyribose ring brea kdown. Both results are consistent with 5'-hydrogen atom abstraction f or initiation of the site-selective nicking. Hydrogen atom abstraction at other sites of the deoxyribose ring and/or base oxidation and loss followed by strand scission likely are responsible for the nonspecifi c nicking. The 5'-abstraction mechanism contrasts to those elicited by other O-2-derived and metal-associated oxidants, which may provide a biomarker for the reactivity of HOO. in vivo.