Figure 8.

Deficiency in 3′-dephosphorylation of NCS-C-induced DSBs in PNKP^−/−/− HeLa cells as determined by LMPCR. Cells were treated with 5 μM NCS-C and incubated at 37°C for 5-180 min. DNA was isolated and 3′ termini of DSBs were assessed by ligation-mediated PCR. A. Experimental scheme. NCS-C induces DSBs preferentially at AGT●ACT sequences, including at bp 36-38 of the human Alu repeat. The left-hand DSB end has an overhanging 3′-phosphate. To quantify these phosphate ends, any existing 3′-hydroxyl ends are first blocked by tailing with TdT and dTTP/ddTTP. The 3′-phosphates are then removed with CIP and the resulting 3′-hydroxyl DSB ends are ligated to an anchor and quantitated by ligation-mediated real-time PCR. To quantitate 3′-hydroxyl DBSs formed in the cell by PNKP, DNA from NCS-C-treated cells is subjected to LMPCR without any prior enzyme treatments, B. Principle of Taqman PCR. As PCR product accumulates, the fluorescent probe binds to the Alu/anchor junction after each denaturation cycle and the fluorophore is released by the 5′→3′ exonuclease of Taq polymerase as it replicates the product, resulting in an exponential increase in fluorescence as a function of cycle number. C. LMPCR amplification profiles from a representative experiment, showing more 3′-phosphate DSBs and fewer 3′-hydroxyl DSBs in PNKP^−/−/− than in WT HeLa cells. Amplification profiles from duplicate PCR reactions are shown for each condition. D. Time course for formation and disappearance of 3′-phosphate and 3′-hydroxyl NCS-induced DSBs, as determined by LMPCR of cell DNA treated with TdT plus CIP or neither enzyme, respectively. C_T values for DNA treated with TdT only are also shown. Error bars show the mean ± SEM from 4 independent experiments.