Breeding strategy to generate the knockout cohorts.

<p><i>Ku80^+/−</i> and <i>dna-pk_cs</i>^+/− animals are backcrossed for multiple generations to C57Bl6/J-pUR288 (left of dashed line) and FVB (right of dashed line) background. Double heterozygous knock out C57BL6/J male mice are crossed with double heterozygous knock out FVB female mice. Using this strategy all four desired cohorts are generated as F1 hybrids (grey boxes).</p

Histopathology neoplastic lesions.

<p>Mentioned n-numbers are tumor bearing animals available for microscopical examination.</p><p><i>k</i>-/-;<i>d</i>-/-: <i>ku80</i>-/-; <i>dna-pk_cs^−/−</i>.</p><p>mes. lm. nd.: mesenterial lymph node.</p><p>ax. lm. nd.: axillary lymph node.</p><p>*∼5 thymus tumor preparations were microscopically examined per group and found to be all lymphomas. All other macroscopic neoplastic lesions observed in thymus are assumed to be lymphomas as well.</p><p>** analyzed in femur.</p><p>Tumor incidence based on all animals from longevity cohort, including those not microscopically examined.</p

Mean body weights.

<p>(A) Males. (B) Females.</p

Survival curves.

<p>Median survivals are shown in the figure legends in parentheses. <i>dna-pk_cs^−/−</i> mice live significantly longer compared to <i>ku80^−/−</i> mice in both male (<i>p</i> = 0.01) and females (<i>p</i> = 8.7*10⁻⁶) mice. Survival of double knock out mice is identical to <i>ku80^−/−</i>. (A) Males. (B) Females.</p

Histopathology non-neoplastic lesions.

<p>4–5 animals examined per group. All selected mutant mice are at end of life, wild type mice are aged matched to mutant mice.</p><p>Mean age in weeks is shown (w).</p><p>mes ln: mesenterial lymph node.</p><p><i>k</i>-/-;<i>d</i>-/-: <i>ku80</i>^−/−; <i>dna-pk_cs^−/−</i>.</p><p>St. Dev.: Standard deviation.</p><p><i>p</i>-values based on comparison with wild type animals.</p><p>* <i>p</i><0.05.</p><p>** <i>p</i><0.01.</p><p>*** <i>p</i><0.001.</p

Molecular beacon assay to measure APE1 activity.

<p>No Ku70 was compared to Ku70 added to substrate with or without APE1. Fluorescence: the excitation wavelength is 485 nm and the emission wavelength is 538 nm. Shown is the average of three experiments with error bars (standard deviation). (A) Ku70^1–609 (full-length Ku70) (B) Ku70^115–609. (C) Ku70^1–115. (D) Ku70^1–300.</p

<i>In vivo</i> analysis. Life span for <i>ku80^-/-</i> and <i>dna-pk_cs^-/-</i> (A) males and (B) females (45 mice in each cohort).

<p>Mutation spectrum in <i>ku70^-/-</i> mice with and without p53 in the (C) liver and (D) brain. Size changes are chromosomal rearrangements that include translocations and large insertions/deletions. No size changes are point mutations (base changes and small insertions/deletions). A student t test was performed for a statistical analysis and tables are presented showing all possible comparisons.</p

CRT0044876 (CRT) survival fraction (SF).

<p>All cells are deleted for p53 (even controls) to avoid early replicative senescence. Shown is the average of three experiments. (A) Cells deleted for Ku80 are hypersensitive to CRT. Expression of APE1 or mouse Ku80 rescued CRT hypersensitivity for Ku80-mutant cells. (B) Cells deleted for either Ku70 or Ku80 but not both were hypersensitive to CRT demonstrating independent function for the individual Ku subunits as opposed to the Ku heterodimer. (C) Cells deleted for Ku70 or Ku80 or both were hypersensitive to γ-radiation demonstrating the Ku heterodimer repaired damage as opposed to independent function for the individual proteins.</p

Ku70 and Ku80 bind to AP sites.

<p>We show 10% input for <i>in vitro</i> translated product. The same concentration of biotinylated substrate were used for each reaction of the binding assays. Right panel shows the relative band intensity as measured with the Kodak document program and as compared to 10% input. Shown is the average of three experiments with error bars (standard deviation). Statistics are shown in the results. (A) Ku70 and Ku80, but not Ku70+Ku80, preferentially bind to the AP site. (B) GzmA cleaves Ku70 at Arg(301). Cleavage at this site separates the two Ku80 binding domains. The GzmA N-terminal Ku70 cleavage product (Ku70^1–300) binding and competition assays. (C) Ku70^1–115, but not Ku70^115-609, bound to the AP/G substrate at a higher level than the C/G and U/G substrates.</p

Epistatic analysis for Ku80 and Pol β.

<p>All cells are deleted for p53 (even controls) to avoid early replicative senescence. Shown is the average of three experiments. (A) Western showing RNAi knockdown of Pol β in <i>p53^-/-</i> control and <i>ku80^-/- p53^-/-</i> fibroblasts that stably express a shRNA plasmid specific for mouse Pol β (three clones). PCNA was loaded to normalize for nuclear protein levels. The expression of endogenous Pol β in the shRNA-transduced cell lines was undetectable by immunoblot, consistent with our earlier reports for this mouse Pol β -specific shRNA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086358#pone.0086358-Trivedi1" target="_blank">[30]</a>. (B) Dose-response to MMS for <i>p53^-/-</i> control and <i>ku80^-/- p53^-/-</i> fibroblasts with and without mouse Pol β shRNA expression. (C) Western showing increased Pol β levels for the <i>ku80^-/- p53^-/-</i> fibroblasts that stably express a Pol β expression plasmid (two clones). Beta-actin was loaded to normalize for cellular protein levels. (D) Pol β -overexpression rescues Ku80-mutant phenotype for MMS.</p