Figure 2
N-terminal domain is essential for in vivo Tdp1 catalysis of TOP1cc. (a) top1Δ, tdp1Δ cells were co-transformed with vector control (top1Δ) or YCpGAL1-TOP1 U (Top1) and control vector (tdp1Δ) or the indicated YCpGAL1-TDP1 L plasmid. Exponentially growing cultures were diluted to an OD600 of 0.3 and three times ten-fold serially (10–1, 10–2, 10–3, represented by triangle) diluted and spotted onto selective galactose plates, (a) without or (b) 5 with μg/ml camptothecin (CPT). Plates were incubated at 30 °C for 4 days. (c) Yeast transformants from (a-with Top1) grown in selective media supplemented with 2% raffinose were diluted to OD600 of 0.01 in selective media supplemented with 2% galactose and grown for 4 days at 30 °C. Every 24 h OD600 were determined from each culture. Average values of 4 independent growths are shown, SD is not shown for clarity with only Day 3 and Day 4 showing significant difference in 2-tail unpaired student T-test mutant versus tdp1Δ (vector control) for tdp1Δ-H182A (*D3: p = 2.5 10–7; **D4: p = 2.2 10–5) and tdp1Δ-H432N (#D3: p = 1.9 10–8; ##D4: p = 6.3 10–4). (d, e) Ectopically expressed TDP1 protein levels in 20 μg/lane total cell extracts (d) or nuclear extracts (e) from galactose induced transformants used in (a-vector control) resolved on 10% SDS-PAGE and stained with anti-TDP1, stripped and stain with anti-GPDH (d) or anti-Histone H3 (e). (f) Detection of endogenous TDP1 protein in 100 μg/lane of wild-type yeast total cell extracts cultured in dextrose or galactose supplemented media were resolved on 10% SDS-PAGE and stained with anti-TDP1, stripped and stain with anti-GPDH. FL: full-length TDP1; ΔN: N-terminal truncated TDP1 proteins. #: non-specific anti-Tdp1 antibody reactions; Molecular weight marker ladder in KDa. Shown are a representative agar plates (white box added for clear separation of the vector control (tdp1Δ) and Top1 expressing (Top1) spots) and immunoblots, cropped after adjustment of intensity of the whole plate/membrane using photoshop. All images shown are from one membrane or agar plate and are not merged products of different plate or membranes. Full membranes of immunoblots can be found in Fig. S1.
