Figure 2.

Avian Tdp2 repairs Top1-mediated DNA damage in absence of Tdp1. (a) Schematic representation of the wild type and targeted chicken Tdp1 locus. Closed boxes indicate the exons. ‘Hygro’ is a hygromycin cassette and ‘Puro’ is a puromycin cassette. The schematic representation of the targeting locus of the chicken Tdp2 gene has been described previously (13). (b) Genomic DNA from DT40 cells of the indicated genotype was digested with NcoI and Southern blotting was conducted using the probe depicted in (a) (for Tdp1, top) or as described in ref 13 (for Tdp2, bottom). (c) Total cell lysate (2, 10 and 20 μg protein) from avian cells of the indicated genotype was incubated with a nicked duplex DNA substrate harbouring a 3′-phosphotyrosine (‘18-Y’) terminus (top) for 1 h at 37°C and reaction products were fractionated by denaturing PAGE and detected by phosphorimaging. The positions of oligonucleotide substrate (‘18-Y’) and product (‘P-20’) harbouring 3′-phosphotyrosine and 3′-phosphate termini, respectively, are shown. (d) DT40 cells of the indicated genotype were incubated with DMSO or 20 μM CPT for 60 min at 37°C and DNA strand breakage quantified by alkaline comet assays. Mean tail moments were quantified for 50 cells/sample/experiment and data are the average of n = 3 biological replicates ± s.e.m.