Collaboration of homologous recombination and nonhomologous end-joining factors for the survival and integrity of mice and cells

Abstract

Homologous recombination (HR) and nonhomologous end-joining (NHEJ) are mechanistically distinct DNA repair pathways that contribute substantially to double-strand break (DSB) repair in mammalian cells. We have combined mutations in factors from both repair pathways, the HR protein Rad54 and the DNA-end-binding factor Ku80, which has a role in NHEJ. Rad54(-/-)Ku80(-/-) mice were severely compromised in their survival, such that fewer double mutants were born than expected, and only a small proportion of those born reached adulthood. However, double-mutant mice died at lower frequency from tumors than Ku80 single mutant mice, likely as a result of rapid demise at a young age from other causes. When challenged with an exogenous DNA damaging agent, ionizing radiation, double-mutant mice were exquisitely sensitive to low doses. Tissues and cells from double-mutant mice also showed indications of spontaneous DNA damage. Testes from some Rad54(-/-)Ku80(-/-) mice displayed enhanced apoptosis and reduced sperm production, and embryonic fibroblasts from Rad54(-/-)Ku80(-/-) animals accumulated foci of gamma-H2AX, a marker for DSBs. The substantially increased DNA damage response in the double mutants implies a cooperation of the two DSB repair pathways for survival and genomic integrity in the animal.

Figure 1.

Impaired postnatal survival of Rad54^-/-Ku80^-/- mice. The total number of mice for each genotype (n) is indicated. (A) Survival of mice during fostering. Curves begin at birth. Total numbers at birth include animals that were stillborn. Percentage of mice still alive at weaning (i.e., 21 d of age) is indicated at right. (B) Survival of mice after weaning from foster mothers. Curves begin with mice that were alive at weaning (i.e., 21 d of age). As a reference, a survival curve of Rad54^-/- mice that were nursed by their birth mother is depicted. Percentage of weaned mice still alive at 3 or 7 mo of age is indicated; in parentheses, the percentage of mice still alive at 3 or 7 mo relative to the total number of mice recovered at birth is also indicated.

Figure 2.

Growth retardation of Ku80^-/- mice, regardless of Rad54 genotype. Males (A) and females (B) were weighed once a week starting 10 d after birth. Average weights of control mice (i.e., wild-type mice or mice heterozygote for either Rad54 or Ku80 mutation, or both) and Rad54^-/-, Ku80^-/-, Rad54^+/-Ku80^-/-, or Rad54^-/-Ku80^-/- mice are plotted against days after birth. The total number of mice for each genotype (n) is indicated. Because some mice died or were sacrificed during the observation period for other analyses, the number of mice remaining at the end of the study is reduced and is indicated in parentheses. Gray areas indicate weight variations based on standard deviations.

Figure 3.

Ionizing radiation sensitivity of Rad54^-/-Ku80^-/- mice. (A) Survival after 100 cGy γ-irradiation. Each curve represents six mice. Controls are two mice each Rad54^-/-, Rad54^+/-Ku80^+/-, and wild-type. (B) Survival after 200 cGy γ-irradiation. Each curve represents eight mice, except the controls, which are the same as in A.

Figure 4.

Accumulation of γ-H2AX foci in Rad54^-/-Ku80^-/- cells. (A) Immunofluorescence detection of γ-H2AX foci in passage-2 MEFs of indicated genotypes. (B) Fraction of cells with γ-H2AX foci (light-green bars) or no foci (white bars) is shown for each cell line of indicated genotype. Rad54^-/-Ku80^+/x refers to combined results from Rad54^-/-Ku80^+/+ and Rad54^-/-Ku80^+/- MEFs. Counts represent averages from two independent experiments. (C) Fraction of cells exhibiting 0 (white bars), 1-5 (gray bars), 6-10 (light-green bars), or >10 (dark-green bars) γ-H2AX foci per nucleus. Counts represent averages from two independent experiments. (D) Immunufluorescence detection of cleaved caspase-3 (αCaspase-3) in UV-irradiated wild-type or p53^-/- MEFs and in untreated wild-type, Rad54^-/-Ku80^+/-, Ku80^-/x, or Rad54^-/-Ku80^-/- MEFs. Fraction of αCaspase-3-positive cells are shown and represent averages from two independent experiments.

Figure 5.

Increased cell death in Rad54^-/-Ku80^-/- testes. (A) Testis and epididymis sections stained with TUNEL/PAS and DAPI, respectively. In Rad54^+/-Ku80^+/- (panel i), Ku80^-/- (panels ii,iii), and Rad54^-/-Ku80^+/- (panel v) mice, TUNEL-positive testicular cells are rare, but when present, are usually located at the base of the seminiferous tubule (e.g., arrowhead in panel iii). Similar results were obtained for one Rad54^-/-Ku80^-/- mouse (mouse #1; data not shown). In two other Rad54^-/-Ku80^-/- mice, mice #2 (panels vi,vii) and #3 (panels ix,x), TUNEL-positive cells are abundant. In mouse #2, tubules with reduced cellularity and germ-cell depletion (arrowhead in panel vi) are evident. In mouse #3, cellularity of tubules is not as visibly reduced, but TUNEL-positive cells are even more abundant, including round spermatids (arrowhead in panel x). Mature sperm in the epididymis is abundant in all controls, including Ku80^-/- mice (panel iv) and Rad54^-/-Ku80^-/- mouse #1 (data not shown), whereas it is significantly reduced for both Rad54^-/-Ku80^-/- mice #2 (panel viii) and #3 (panel xi). Magnification: panels i,ii,v,vi,ix, 100×; panels iii,vii,x, 400×; panels iv,viii,xi, 200×. (B) Percentages of tubules exhibiting 0 or 1 (white bars), 2-4 (gray bars), 5-10 (light-green bars), or >10 (dark-green bars) TUNEL-positive cells. Counts represent averages from three mice, except for Rad54^-/-Ku80^-/- mice, which are individually depicted. (C) Percentages of tubules exhibiting two or more TUNEL-positive cells. Counts represent averages from three mice.

Figure 6.

Relative contribution of NHEJ and HR in repair of DSBs. See text for details.