Figure 1
Characterization of the nuclease activity of HAN. (a) Schematic representation of the classification of the Cdc45/RecJ family members. Reported exonuclease activities and their directions are shown. TkoGAN, PfRecJ, TaRecJ1 and RecJ2, and MjRecJ1 and RecJ2 are RecJ-like proteins in T. kodakarensis, P. furiosus, T. acidophilum, and M. jannaschii, respectively. (b) Purified recombinant HAN-WT (2 μg) and HAN-D366A (2 μg) proteins were subjected to SDS -10% PAGE followed by CBB staining. Protein size markers were run in lane M, and their sizes are indicated on the left of the gels. (c) Detection of the cleavage direction of HAN for ssDNA. HAN (WT and D366A mutant) (5 nM each) was incubated with 5′-32P-labeled 5 nM ssDNA (dA30) at 70 °C for 1, 2, and 5 min. The 30-nt ssDNAs with or without four successive phosphorothioate modifications from the 10th to 14th bases were used for the substrates and indicated as “normal” (dA30, lanes 1–5) and “S” (dA30ssss14, lanes 6–10), respectively. Lane 11 shows the size marker DNAs (14, 10, and 1 nt, respectively). (d) HAN exhibited 3′ to 5′ exonuclease activity for RNA. GAN and HAN (WT and D366A) (5 nM each) were incubated with 5′- or 3′-FITC-labeled 10 nM RNA at 80 °C for 0.5, 3, and 5 min. HAN cleaved RNA processively (lanes 2 and 3). Substrates in these assays were 5′-FITC-labeled “normal” RNA (FITCrA30, lanes 1–5), 5′-FITC-labeled phosphorothioated (“S”) RNA (FITCrA30ssss14, lanes 6–8), and 3′-FITC-labeled RNA (rA30FITC, lanes 10–11). Products were subjected to 8 M urea-15% PAGE. Lane 9 shows 5′ FITC-labeled RNA (20 nt and 14nt) and DNA (1 nt) size markers.
