Development of a methodology for estimating the degree of localization of AP-sites on DNA with an application of Frster resonance energy transfer
赤松 憲; 鹿園 直哉
Akamatsu, Ken; Shikazono, Naoya
DNA lesions induced by ionizing radiation and chemicals can cause mutation and carcinogenesis. In particular, "clustered damage" site, that is a DNA region with multiple lesions within a few helical turns, is believed to hardly be repaired. This type of damage is considered to be induced around high-LET radiation tracks. However, chemical and spatial details of them are not known. Therefore, we have developed a methodology for estimating the degree of localization of the lesions using Frster resonance energy transfer (FRET). First, we prepared pUC19 with APsites (AP-DNA) by heating in acidic buffer as a randomly-distributed AP-DNA model. The AP-DNA was labeled both with Alexa Fluor 350 and 488 fluorescent dyes (donor and acceptor) with -O-NH groups. The FRET efficiency (E) was calculated using the donor intensities before/after enzymatic digestion of the labeled AP-DNA. Moreover, we have constructed a theoretical equation to obtain E as functions both of AP-site density and of labeling ratio of acceptor to total AP-sites. As a result, we found that experimentally-obtained E values for the heat-treated AP-DNA correspond to theoretical ones calculated on the basis of exponential distribution. Now we are applying the FRET methodology to the DNA irradiated with radiations such as Co -rays and helium ion beam.