Abasic sites preferentially form at regions undergoing DNA replication

Abstract

We investigated whether apurinic/apyrimidinic (AP/abasic) sites were more frequent in regions of DNA replication in cells and whether their number increased during oxidative stress. DNA fiber spreading and fluorescent immunostaining were used to detect areas of DNA replication and sites of AP lesions in extended DNA fibers. The distribution of AP sites was determined in DNA fibers from vertebrate cells maintained under normal culture conditions or stressed with exogenous H(2)O(2). AP lesions per unit length were enumerated in bulk DNA or at replication sites. The background density of AP sites in DNA fibers was 5.4 AP sites/10(6) nt, while newly replicated DNA contained 12.9 AP sites/10(6) nt. In cells exposed to 20 μM H(2)O(2), AP sites in newly replicated DNA increased to 20.8/10(6) nt. Determinations of AP site density in bulk DNA by fiber analysis or standard slot blot assays agreed to within 10%. Our findings show that the fiber assay not only accurately determines the frequency of AP sites but also shows their distribution. They also reveal that there is increased susceptibility to oxidative damage in DNA regions undergoing replication, which may explain the previously observed clustering of AP sites.

Figure 1.

Detection of AP sites in DNA fiber spreads. Composite image of DNA fibers stained with YOYO-1 green fluorescent dye. AP sites (white arrows) were tagged with biotin using ARP (see Materials and Methods) and detected with a red fluorescent anti-biotin antibody. Scale bar = 96.6 kbp.

Figure 2.

Number of AP sites in DNA from cells under typical tissue culture conditions or exposed to 20 μM H₂O₂. Number of AP sites per 10⁶ nt as determined by slot-blot (left) and fiber-spread analysis (right) for cells under normal culture conditions and after exposure to 20 μM hydrogen peroxide. Average values are listed at top. Slot blot average was determined from 3 independent experiments; fiber analysis values were determined from analysis of 6 different slides.

Figure 3.

Number of AP sites in areas undergoing replication. Comparison between average number of AP sites found in replicating DNA fibers before and after exposure to 20 μM H₂O₂. In these experiments, only DNA fibers with replicating DNA were quantified. Average values are listed at top. Fiber analysis values were determined from analysis of 3 slides.

Figure 4.

Detection of AP sites in areas undergoing replication in DNA fiber spreads. Composite image of multiple DNA fibers containing AP sites and areas undergoing replication. Fiber spreads were prepared from cells that were pulsed with IdU (red fluorescence) for 10 min, exposed to H₂O₂, and then pulsed with CldU (green fluorescence) for 20 min. IdU and CldU were identified as described in Materials and Methods. AP sites were tagged with biotin using ARP, and biotin was identified using a blue fluorescent antibody. For ease of viewing, the blue signal corresponding to AP sites was electronically changed to white. Scale bar = 60 kbp.