Figure 6.

Exonuclease activity of TTHB178 against various DNA structures. (A–C) The 3′-overhanging (50sf + 40sr) (A), Y structure (50sf + 20sr) (B) and gapped flap structure (50sf + 21sr + 28sr) (C) DNAs were reacted with 3-μM TTHB178 for various reaction periods. The reaction time is indicated at the top of the panels. Assays for the Y structure and gapped flap structure were carried out at 20°C to stabilize the short dsDNA region of the substrates. As TTHB178 showed relatively weak activity at 20°C compared with that at 37°C or 60°C, the assays were performed for a prolonged reaction time. The assay for 3′-overhanging DNA was carried out at 37°C. ‘C’ means the substrate incubated without TTHB178 for 27 h. (D) Activity for an ssDNA with a 3′-H terminus. The substrate 21-mer ssDNA (21r) was 3′-end-labelled with [α-³²P]cordycepin-5′-triphosphate and reacted with 3-μM TTHB178 at 37°C. The reaction time is indicated at the top of the panel. ‘C’ means the substrate incubated without TTHB178 for 30 min. In all the panels, the digested products were analysed by electrophoresis through denaturing 8 and 25% polyacrylamide gels. ‘M’ means the 40- (in A) and 19-mer (in B and C) of marker DNAs. The bands seen near the bottom of the gels in B and C might be experimental artefacts because they were not observed reproducibly.