Figure 1.

The dual incision activity of human nucleotide excision repair on duplex plasmid DNA containing a single ribonucleotide. (a) The DNA substrate used for the in vitro dual incision assay. The substrate plasmid harbored a single 6-4PP or dG, 8-oxo-dG, rG, or 8-oxo-rG at a defined position in the DNA sequence between PstI and HindIII sites of pBluescript II KS(+) as previously described⁵³. (b) In vitro dual incision assays were performed using purified NER proteins and internally ³²P-labeled DNA substrates containing a 6-4PP, dG, 8-oxo-dG, rG, or 8-oxo-rG as described in the Methods. DNA samples were subjected to 10% denaturing polyacrylamide gel electrophoresis followed by autoradiography. The full-length gel is shown in Supplementary Fig. S1. (c) Quantification of the products formed by NER-mediated dual incision reaction. Values are presented as mean ± S.E. of three independent experiments. Significant differences are indicated by asterisks (**P < 0.01, ****P < 0.0001 by two-way ANOVA).