**4.2 HCR for HR repair**

472 Selected Topics in DNA Repair

3. The supernatants (50 l, adjustable) are transferred into a 96-well plate and 20 l of

4. Ten minutes later, the firefly luminescence is measured by a microplate luminometer

5. Add 20 l of Dual-Glo™ Stop & Glo® Reagent (Promega) to each well and wait for 10

6. The transfection efficiency-adjusted firefly luciferase activity is obtained by dividing the

Since the pCMV-Luc is damaged by UV, the DNA repair activity (responsible to UV) can be represented as the *Renilla*–calibrated firefly luciferase activity derived from UV-damaged pCMV-Luc verse to those from undamaged pCMV-Luc. By this way, one can compare the effects of various environmental toxicants on cellular DNA repair capacity. For example, an inhibitory effect of arecoline on the repair of UV-damaged pCMV-Luc can be found by

(C) Comparison of the effects of arecoline (ARE, 0.3 mM) and its vehicle (distilled water) on

Fig. 1. Schematic illustration of host cell reactivation (HCR) assay for examination of

(A) The reporter plasmid pCMV-Luc is prepared in *E. coli*. (B) The pCMV-Luc is damaged by 1000 J/m2 of UV light.

the repair of UV-damaged pCMV-Luc in HEp-2 cells.

Dual-Glo™ Luciferase Reagent (Promega) are added to each well.

(Centro LB 960, Berthold, Bad Wildbad, Germany).

minutes, then the *Renilla* luminescence is read.

**4.1.5 Representation of NER activity by HCR assay** 

*Renilla* luciferase activity.

using HCR assay (Fig. 1C).

nucleotide excision repair.

HR is a reliable mechanism to accurately repair DNA double strand breaks. Here we use PCR to generate two overlapping DNA fragments that contain i) CMV IE promoter and 5' part of *Renilla* luciferase gene, ii) 3'-part of *Renilla* luciferase gene and poly-A tail sequence from pRL-CMV (Fig. 2A) and serve as substrates for HR (Fig. 2B). The two overlapping DNA fragments can also be produced by restriction enzyme digestion and gel elution.
