A method for detecting abasic sites in living cells: age-dependent changes in base excision repair

Abstract

Apurinic/apyrimidinic (AP) sites are common DNA lesions that arise from spontaneous depurination or by base excision repair (BER) of modified bases. A biotin-containing aldehyde-reactive probe (ARP) [Kubo, K., Ide, H., Wallace, S. S. & Kow, Y. W. (1992) Biochemistry 31, 3703-3708] is used to measure AP sites in living cells. ARP penetrates the plasma membrane of cells and reacts with AP sites in DNA to form a stable ARP-DNA adduct. The DNA is isolated and treated with avidin-horseradish peroxidase (HRP), forming a DNA-HRP complex at each biotin residue, which is rapidly separated from free avidin-HRP by selective precipitation with a DNA precipitating dye (DAPER). The number of AP sites is estimated by HRP activity toward chromogenic substrate in an ELISA assay. The assay integrates the AP sites formed by the different glycosylases of BER during a 1-h incubation and eliminates artifactual depurination or loss of AP sites during DNA isolation. The assay was applied to living cells and nuclei. The number of AP sites after a 1-h incubation in old IMR90 cells was about two to three times higher than that in young cells, and the number in human leukocytes from old donors was about seven times that in young donors. The repair of AP sites was slower in senescent compared with young IMR90 cells. An age-dependent decline is shown in the activity of the glycosylase that removes methylated bases in IMR90 cells and in human leukocytes. The decline in excision of methylated bases from DNA suggests an age-dependent decline in 3-methyladenine DNA glycosylase, a BER enzyme responsible for removing alkylated bases.

Figure 1

Standard curve and background at 450 nm from ARP-derivatized DNA. DNA with AP sites was prepared by incubating uracil-DNA glycosylase with 100 ng of genomic DNA of E. coli that contains a known amount of uracil. The DNA was then treated with ARP to trap AP sites, as described. A DNA sample not treated with ARP was run in parallel as background. Both samples of DNA were incubated with HRP-avidin and prepared as described. (A) Typical standard curve. (B) Shows signal and background.

Figure 2

Kinetics of ARP accumulation into intact IMR90 cells. IMR90 cells (3 × 10⁶/ml) in PBS/5 mM glucose were incubated with 3 mM ARP at 37°C for different intervals. At each time point, cells were sampled and processed as described in Materials and Methods, and the intracellular ARP was measured by HPLC-EC. The data is an average of three independent experiments (mean ± SD).

Figure 3

Endogenous rate of AP sites formation in intact IMR90 cells. Senescent and young IMR90s (2 × 10⁶/ml) in PBS/5 mM glucose were treated for different intervals with 5 mM ARP at 37°C. Then, free ARP was washed out followed by isolation of DNA, and the level of AP sites was determined as described in Materials and Methods. The values are mean ± SE. *, P < 0.05; **, P < 0.01.