DNA-dependent protein kinase suppresses double-strand break-induced and spontaneous homologous recombination

Abstract

DNA-dependent protein kinase (DNA-PK), composed of Ku70, Ku80, and the catalytic subunit (DNA-PKcs), is involved in repairing double-strand breaks (DSBs) by nonhomologous end-joining (NHEJ). Certain proteins involved in NHEJ are also involved in DSB repair by homologous recombination (HR). To test the effects of DNA-PKcs on DSB-induced HR, we integrated neo direct repeat HR substrates carrying the I-SceI recognition sequence into DNA-PKcs-defective Chinese hamster ovary (V3) cells. The DNA-PKcs defect was complemented with a human DNA-PKcs cDNA. DSB-induced HR frequencies were 1.5- to 3-fold lower with DNA-PKcs complementation. In complemented and uncomplemented strains, all products arose by gene conversion without associated crossover, and average conversion tract lengths were similar. Suppression of DSB-induced HR in complemented cells probably reflects restoration of NHEJ, consistent with competition between HR and NHEJ during DSB repair. Interestingly, spontaneous HR rates were 1.6- to >3.5-fold lower with DNA-PKcs complementation. DNA-PKcs may suppress spontaneous HR through NHEJ of spontaneous DSBs, perhaps at stalled or blocked replication forks. Because replication protein A (RPA) is involved in both replication and HR, and is phosphorylated by DNA-PKcs, it is possible that the suppression of spontaneous HR by DNA-PKcs reflects regulation of replication-dependent HR by DNA-PKcs, perhaps by means of phosphorylation of RPA.

Figure 1

HR substrates. (Upper) Two copies of a 1.4-kbp fragment carrying the neo gene (open and hatched boxes) are separated by a simian virus 40 (SV40) promoter-driven gpt gene. The upstream neo is driven by the MMTV promoter, and is inactivated by a frameshift insertion consisting of an I-SceI site. The downstream copy (neo12) is inactive because it lacks a promoter. neo12 contains 12 silent, single-base mutations that create RFLPs for mapping conversion tracts (for details, see ref. 28). (Lower) HR substrate in which the downstream neo lacks RFLPs.

Figure 2

Functional complementation with human DNA-PKcs. Cells were irradiated with ¹³⁷Cs γ-rays and the resulting colonies with at least 50 cells were counted. Data are averages of three determinations per strain. Control data (AA8 and V3) are reproduced in each panel.

Figure 3

Western analysis of DNA-PKcs in wild-type (AA8), mutant (V), and complemented (-C) cells. Detection with anti-DNA-PKcs antibodies is shown above; anti-actin loading controls run in parallel are shown below.

Figure 4

DSB-induced HR is suppressed by DNA-PKcs. Average HR frequencies (±SD) are shown for DNA-PKcs defective and complemented cell lines for 3–6 determinations per cell line. Value for each complemented derivative is significantly different from respective parent line (*, P < 0.01; **, P < 0.001; t tests). Hatched bars represent average HR frequencies of three independent pPur (control) transfectants of V24, V719, or VD13, based on three determinations per transfectant. None of the pPur values are significantly different from respective parent lines (all P > 0.15, t tests).

Figure 5

Spontaneous HR is suppressed by DNA-PKcs. HR rates for V24-C2 and V714-C1 represent minimum estimates because the median number of G418-resistant colonies was zero. Fold decreases with DNA-PKcs complementation are given above bars.

Figure 6

Potential roles of DNA-PKcs in repair of replication-dependent DSBs or lesion bypass. (A) Nicks are converted to DSBs when encountered by a replication fork. DNA-PKcs might facilitate direct ligation of single-stranded ends, or it might transiently bridge the discontinuity, allowing replication to proceed. (B) During recombinational lesion bypass, the nascent end at a blocked replication fork invades the sister chromatid. If repeats are present (shown by thick segments), invasion can occur in misaligned or correctly aligned modes, producing recombinants or nonrecombinants, respectively. In either case, reassociation of this strand with the original template effects lesion bypass.