**4.2.2 Substrate preparation for HR repair**

1. Set up PCR reaction as below:



Alternative: the two DNA fragments used for HR can also be generated by using a combination of restriction endonucleases *Bgl*II/*Nhe*I and *Pst*I/*Bam*HI for pRL-CMV (Progema). The use of former restriction enzymes will produce a DNA fragment containing

Application of Host Cell Reactivation in Evaluating the Effects of Anticancer Drugs

formation of DNA-LipofectamineTM 2000 complexes.

experimental design.

4. Perform dual-luciferase assay as section 4.1.4.

extracts on homologous recombination repair.

precise and overall NHEJ repair activities, respectively.

**4.3 HCR for NHEJ repair** 

and Environmental Toxicants on Cellular DNA Repair Activity in Head and Neck Cancer 475

3. Add the 100 μl of DNA-LipofectamineTM 2000 complexes to each well and incubate at 37°C in a CO2 incubator for 6 h. Then the cells can be treated with toxicants (such as areca nut extracts, ANE) or anticancer drugs for another 24 h. Note: cells can be treated with toxicants or anticancer drugs prior to transfection that is dependent on

5. Determine the HR activity by comparing the firefly luciferase-calibrated *Renilla* luciferase activities between the environmental toxicants- or anticancer drugs-treated cells and vehicle-treated control cells. For examples, the treatment of anticancer drug camptothecin (CPT, a topoisomerase I inhibitor) can potentiate HR repair activity but

the areca nut extracts (ANE) repress HR repair in HEp-2 cells (Fig. 3).

Fig. 3. The use of HCR assays in evaluating the effects of camptothecin and areca nut

(A) Using the method illustrated in Fig. 2D, the *Renilla* luciferase activity (reflecting the HCR activity in Y-axis) can only be detected in the presence of both two DNA fragments (N+P, lane 3) but not in cells transfected with only one fragment (NheI or PstI, lanes 1 and 2). Treatment of camptothecin (CPT) stimulates HR repair efficiency in the cells (lane 4). (B) Dose-dependent repression of HR repair activity by areca nut extracts (ANE).

NHEJ is another DNA repair mechanism responsible to DSB. Unlike HR repair using sisterchromatids as templates, NHEJ directly joins the broken DNA ends by trimming a few nucleotides on the ends. Therefore, it is thought as an error-prone repair system. In this regard, we prepare two kinds of reporter DNA substrates that are suitable for analyzing the

For overall NHEJ repair, pRL-CMV is linearized with *Hin*dIII that cuts the flanking sequence between CMV promoter and the Renilla luciferase coding sequence. The luciferase will express after re-ligation regardless the loss of some nucleotides. For examining precise

c. Combine the diluted DNA with the diluted LipofectamineTM 2000 (total volume is 100 μl). Mix gently and incubate for 20 min at room temperature to allow the

CMV IE promoter and 5'-part of *Renilla* luciferase gene, the later ones result in 3'-part of *Renilla* luciferase gene and the poly-A signal. These two DNA fragments contain a 222-bp overlapping region for recombination (Fig. 2D).

Fig. 2. Schematic representation of substrate preparation for HCR assay of homologous recombination (HR) repair.

(A) Location of PCR primers on the pRL-CMV reporter plasmid.

