**4.3 HCR for NHEJ repair**

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 precise and overall NHEJ repair activities, respectively.

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

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

**6. Acknowledgment** 

pp. 12985-12990.

Vol. 434, No.7035, pp. 864-870.

**7. References** 

and Environmental Toxicants on Cellular DNA Repair Activity in Head and Neck Cancer 477

modulate cellular response to DNA-damaging anticancer drugs, alterations of DNA repair genes may be involved in the development of resistance to chemotherapy and radiotherapy. In addition, DNA repair activity plays an important role in preventing the mutagenicity and cytotoxicity induced by numerous environmental carcinogens and toxicants. Cells with reduced DNA repair activity may thus be prone to pathological transformation. Therefore, examining the DNA repair activity of a cell can help us to understand the probability of cellular tumorigenicity associated with exposure of environmental carcinogens and is able to assess the responses of various regimens of anticancer treatment. HCR assay is an easy and fast functional assay that can be applied to investigate several DNA repair pathways and is one of the most useful methods for evaluating cellular DNA repair activity *in vivo*.

The authors thank the financial support (grant No. NSC97-2311-B-037-002-MY3 to C.S.L. and NSC99-2314-B-309-004-MY2 to J.L.H.) from the National Science Council, Taiwan.

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NHEJ, *Afl* III that digests the coding region of *Renilla* luciferase gene is used and the luciferase can only be expressed after exact repair (Fig. 4A). The linearized reporter DNA fragments are purified, transfected into host cells and examined for luciferase activity as described above. Below is an example of evaluating the effect of areca nut extracts on precise and overall NHEJ repair (Fig. 4B).

Fig. 4. HCR assay for non-homologous end-joining (NHEJ) repair. (A) The *Afl* III-digested pRL-CMV is used as a substrate for analyzing precise NHEJ repair activity because of the need of exact joining of the *Renilla* luciferase coding sequence. For overall NHEJ, *Hin*d III that cuts the flanking sequence between CMV promoter and the *Renilla* luciferase gene is used. The expression of luciferase is not affected by loss of a few nucleotides in this region during the end-joining process.

(B) The effect of areca nut extracts (ANE, 800 mg/ml for 24 h) on precise (left panel) and overall (right panel) NHEJ repair.
