Figure 6. NER cleavage reaction products generated using DNA templates with 3′ end-labeled rNMP-containing strands.

I; Cartoon of the synthetic substrates used in the in vitro assays, with the sites of incision and expected product size indicated, along with the DNA sequence containing rNMP(s) and mismatched nucleotides. II; The 50-mer duplexes (10 nM) in which the modified strand was 3′ end-labeled (indicated by *), were incubated with the NER proteins at concentrations of 40 nM (UvrA), 200 nM (UvrB), and 100 nM (UvrC) for 60 min at 55°C in the presence of 1 mM ATP. The DNA duplexes DNA-RNA-DNA hybrids either containing a single rAMP (panel A), two consecutive rAMPs (panel B) were assayed. The reaction products were separated under denaturing conditions by 15% polyacrylamide gel electrophoresis (PAGE). The efficiency of UvrABC-dependent incision was determined as a percentage of the radioactivity in the incised (% inc.) products relative to the total signal of the substrate. The data below the gels are mean values calculated from at least two independent experiments. The DNA sequence containing rNMP(s) is shown alongside the gels where DNA and RNA are represented by uppercase and lowercase letters, respectively. The full-sized DNA template (50-mer) and incision products of 19–20 bp are indicated. Orange arrows indicate the cleavage sites. The in vitro assays reveal that the NER proteins incise the DNA backbone 4–5 base pairs 3′ of ribonucleotides and that the efficiency of the reaction is largely unaffected by base-mispairs.