Impact of the polλDN form expression on intrachromosomal NHEJ. (A) Substrate used to measure NHEJ. The only expressed gene is H2-Kd, which is under control of the pCMV promoter. CD8 is not expressed because it is in inverted orientation. CD4 is not expressed because it is too far from the pCMV promoter. Two I-Sce-I sites are present in non-coding sequences. After cleavage by I-Sce-I, the internal H2-Kd/CD8 fragment is excised. Re-joining of the DNA ends can lead to two different measurable events, deletion which leads to expression of the CD4 gene and inversion which leads to expression of the CD8 gene. (B and C) Representation of the sequences of the I-Sce-I restriction sites in the C′10 and A′7H cell lines, respectively. In the C′10 cells the two I-Sce-I sites are in direct orientation whereas in the A′7H cells the two I-Sce-I sites are in inverted orientation (upper panel). Evaluation of the frequency of the deletion (CD4) and inversion (CD8) events in the different cell lines (median panel). Results are the mean +/− SD of three independent experiments. Asterisks represent significant statistical difference (P < 0.05). Western blot for polλ expression in the different cell lines (lower panel).

Sequencing of the repair junctions. (A) Examples of sequences of the repair junctions of the A′7H cell line expressing the different forms of polλ. The structures of the implicated DNA ends are shown at the top of the figure. Black squares, location of the microhomologies. (B) Models for the different classes of end-joining events involving the four protruding nucleotides of the I-Sce-I cleavage site, using microhomology. (C) Percentages of the different types of NHEJ junction in the polλ WT and polλDN expressing cells.

Chromosomal aberrations induced by IR in polλDN expressing cells. Photographs of metaphase spreads of polλDN expressing cells 24 h after treatment with IR at 2 Gy. (b, break; t, triradial).

Effect of the expression of the polβ and polμ inactive form on NHEJ events. (A and B) Representation of the sequences of the I-Sce-I restriction sites in the C′10 and A′7H cell lines, respectively. In the C′10 cells the two I-Sce-I sites are in direct orientation whereas in the A′7H cells the two I-Sce-I sites are in inverted orientation (upper panel). Evaluation of the frequency of the deletion (CD4) and inversion (CD8) events in the different cell lines (median panel). Results are the mean +/− SD of three independent experiments. Western blots for polβ and polμ expression in the different cell lines (lower panel).

Cellular sensitivity to DNA damaging agents of the polλDN expressing cells. (A) Western blot analysis of the expression of the different forms of polλ in the DRA10 cell line. DRA10 is the parental cell line, CT2B is a cell line expressing the empty vector, R2 and R15 are independent cell lines overexpressing the WT form of polλ, RD10 and RD14 are independent cell lines expressing the polλDN form. (B) Cell survival after ionizing radiation and (C) Comparison of cell survival after ionizing radiation of polλDN-transfected cells with the NHEJ-defective cells, XR-1 (xrcc4 mutant cell line) and the XRCC4-complemented cell line (X4V). (D) Homologous recombination activity. (E) Cell survival after treatment with mitomycin C. (F) Cell survival after camptothecin treatment. (G) Topoisomerase I expression in the different polλ expressing cell lines. All cell survival and homologous recombination results are the mean +/− SEM of 3 independent experiments.