Characterization of hydroxyl free radical mediated damage to plasmid pBR322 DNA
Schneider, J.E.; Browning, M.M.; Zhu, X.; Eneff, K.L.; Floyd, R.A.
Mutation Research 214(1): 23-31
1989
ISSN/ISBN: 0027-5107 PMID: 2549408 Document Number: 336818
We have investigated hydroxyl free radical mediated damage to pBR322 DNA produced by ascorbate/iron and oxygen in a phosphate-buffered in vitro system. An observed lag phase in DNA nicking suggest a multi-target model of hydroxyl free radical attack on DNA. In the present report we further examine the model system and show that there is a "heat labile" component of the ascorbate/iron system which can be completely restored by the readdition of ascorbate. These observations have allowed us to rule out the possibility that intermediates build up in the reaction and act independently of ascorbate to increase the reaction rate. We have investigated the initial rate of .ovrhdot.OH production with two .ovrhdot.OH trapping agents, salicylate and deoxyguanosine, and find that the lag in DNA nicking is not due to a corresponding lag in the production of .ovrhdot.OH as assessed by formation of the products, dihydroxybenzoic acids and 8-hydroxydeoxyguanosine, respectively. We have found that the energy of activation for DNA supercoiled nicking is 13.9 kcal/mole and for .ovrhdot.OH trapping by salicylate is 21.1 kcal/nmole. These two activation energies are sufficiently different to suggest that the rate-limiting steps of these two reactions are different. Investigation of the rate of oxygen consumption during the ascorbate/iron-mediated DNA damage showed that oxygen was not a limiting component at any point in the reaction. The addition of catalase slowed down oxygen consumption by 31% and this data taken together with our previous observations on the model implicate hydrogen peroxide as a key intermediate in DNA damage caused by hydroxyl free radical.