Figure 5.

Assays with TTC0482 for uracil processing and AP endonuclease activities. (A) SDS–PAGE analysis of purified TTC0482 and TTUDGA from T. thermophilus HB27 after IMAC and heparin affinity chromatography (for details refer to ‘Materials and Methods’ section). M, molecular weight markers as in Figure 2A. Calculated relative molecular weights for TTC0482 and TTUDGA are 31 200 and 23 680, respectively. (B) Gel electophoretic analysis of reaction products after incubation of substrates (0.12 pmol) with 0.6 pmol of the EndoIV homologue TTC0482 or/and the uracil DNA glycosylase TTUDGA (12) from T. thermophilus HB27 as indicated. The substrates are identical to the ones used for the glycosylase assay of MJ1434 (Figure 3D). NaOH±: with or without post-reaction NaOH-treatment. Note that the AP-endonuclease reaction product shown in lane 7 migrates as a 15-mer with a 3′-OH terminus (compare marker in lane 1). For reaction details refer to ‘Materials and Methods’ section.