Abstract
In a living cell, a multiply damaged site in DNA is thought to be repaired by several different repair pathways simultaneously or sequentially. Under this situation the final cellular response to the lesion cluster might depend on the order of repair processes because the configuration of the lesions will be modified by the reaction of the initial repair protein, affecting the DNA-binding or lesion-excision activities of the latter repair protein. For example, a cluster comprised of an AP site or SSB and base lesions is formed after one of the base lesion in a base lesion cluster is exicesd by a glycosylase protein. Theoretical molecular dynamics simulation study showed that SSB proximately located 8-oxo-guanine could inhibit the binding activity hOGGI (Higuchi et al, 2010). In the present study, we investigate how the initial enzymatic repair affects the activity of the latter repair enzyme. Plasmid DNA (pUC18) irradiated with C6+ion is treated with two base excision repair enzymes, Nth and Fpg, which convert pyrimidine and purine lesions to a SSB. The enzymatic activities are quantified by measuring the conformational changes of the plasmid using agarose gel electrophoresis. Obtained results show that the amount of enzymatically induced SSB is slightly (about 5%) less in DNA sample treated with Nth first and then Fpg than that in the sample treated with Fpg first and then Nth, or with both enzymes simultaneously. The repairability of clustered damage induced by high-LET ions will be discussed.
