FIGURE 2.

Targeted disruption of Tdp1 in DT40 cells and characterization of Tdp1−/− cells. A, schematic representation of the chicken Tdp1 locus and configuration of the targeted allele. Black squares indicate the exons. Two targeting plasmids, Tdp1-1-puro and Tdp1-2-hyg, with different homology arms and resistance genes were used. The restriction enzyme sites used for the Southern blot analysis are indicated. Primers (a–d) used for the RT-PCR in C are indicated by arrows. The probe used in the Southern blot analysis is indicated as a horizontal bar. loxP sites are indicated as black triangles flanking each resistance gene (white square). gg, gallus gallus. B, Southern blot analysis of NcoI- and NotI-digested genomic DNA from cells with the indicated genotypes using a flanking probe as shown in A. The arrowheads indicate the position of the detected band. C, representative RT-PCR analysis with the indicated genotypes using the paired primers shown in A. D, Western blot analysis of whole cell lysates prepared from the indicated genotypes. Blots were probed with anti-human TDP1 and anti-actin. E, growth curves of cells of the indicated genotypes. The error bars represent S.D. (n = 3). F, Tdp1 is the primary 3′-tyrosyl phosphodiesterase in DT40 cells. Top, scheme for the conversion of the 14-nt 3′-phosphotyrosyl substrate (14Y) to the 14-nt 3′-hydroxyl product (14OH) by sequential action of Tdp1 and polynucleotide kinase (PNKP). Bottom left, representative gel images of cleavage assays from the indicated cell lines. 5′-³²P-Labeled 14Y (1 nm) was incubated with serially diluted (1:3) whole cell lysates. The highest concentration was 8.8 μg in a 10-μl reaction volume. Bottom right, quantification of processing activity for each cell lysate.