The alternative end-joining pathway for repair of DNA double-strand breaks requires PARP1 but is not dependent upon microhomologies

Abstract

Non-homologous end-joining (NHEJ), the major repair pathway for DNA double-strand breaks (DSB) in mammalian cells, employs a repertoire of core proteins, the recruitment of which to DSB-ends is Ku-dependent. Lack of either of the core components invariably leads to a repair deficiency. There has been evidence that an alternative end-joining operates in the absence of the core components. We used chromosomal reporter substrates to specifically monitor NHEJ of single I-SceI-induced-DSB for detailed comparison of classical and alternative end-joining. We show that rapid repair of both compatible and non-compatible ends require Ku-protein. In the absence of Ku, cells use a slow but efficient repair mode which experiences increasing sequence-loss with time after DSB induction. Chemical inhibition and PARP1-depletion demonstrated that the alternative end-joining in vivo is completely dependent upon functional PARP1. Furthermore, we show that the requirement for PARP1 depends on the absence of Ku but not on DNA-dependent protein kinase (DNA-PKcs). Extensive sequencing of repair junctions revealed that the alternative rejoining does not require long microhomologies. Together, we show that mammalian cells need Ku for rapid and conservative NHEJ. PARP1-dependent alternative route may partially rescue the deficient repair phenotype presumably at the expense of an enhanced mutation rate.

Figure 1.

Structure of the NHEJ reporter constructs pEJ and pEJ2. GFP translation is prevented by insertion of an artificial ATG (start codon) into the 5′-untranslated region between CMV promoter and the ORF. The artificial ATG is not in frame with the original ATG (17). Two repeat I-SceI recognition sequences (bold characters) flank the artificial ATG either in direct (pEJ2) or in inverted orientation (pEJ). Simultaneous cleavage of both I-SceI sites leads to pop out of the artificial ATG (middle) leaving either non-compatible ends (pEJ) or fully compatible ends (pEJ2). Repair of the I-SceI-induced DSB by NHEJ restores GFP translation leading to green fluorescence.

Figure 2.

Alternative end-joining pathway is efficient but slow for both compatible and non-compatible ends. Ten to thirteen independent clones of CHOK1 and xrs5 cells harboring either pEJ (A) or pEJ2 (B) were transfected with the I-SceI-expressing vector. After a 24-h repair interval the fraction of GFP-positive/total cells (%GFP⁺) was measured by flow cytometry. Hatched columns represent mean values (±SE). xrs5-pEJ clone #11 was not included as deviation from others was larger than 2σ. NHEJ efficiency for both ends and was significantly lower in xrs5 than in CHOK1 cells (Mann–Whitney, P = 0.001). (C) NHEJ kinetics for non-cohesive (left panel) and cohesive (right panel) ends. Cells were harvested at different time intervals after transfection and analyzed for GFP⁺. The mean (±SE) values of three to four representative clones of each variant (CHOK1-pEJ #3, 40, 75; CHOK1-pEJ #33, 39, 48; xrs5pEJ #20, 23, 26, 59; and xrs5-pEJ2 #9, 10, 12) are shown. Differences between the two strains were significant only after 24 h (Mann–Whitney, P = 0.0025).

Figure 3.

Fidelity of the alternative end-joining. After 24 or 48 h of transfection, GFP⁺ cells were sorted and subjected to sequencing (Supplementary Figure S1). Scatter plots show deletion length of all individual repair junctions of (A) non-cohesive (pEJ) or (B) cohesive ends (pEJ2). Deletions are defined as the sum of base pairs lost at both external DSB ends. The 34-bp internal pop-out between both I-SceI sites was not defined as a deletion. P-values indicate the significance level of differences between 24 and 48 h in xrs5 clones (Mann–Whitney test). (C) and (D) Distribution of length of microhomologies used for formation of the repair junctions at both substrates. The 4-nt microhomologies employed by CHO-K1-pEJ2 cells correspond to the unmodified 4-bp complementary overhangs created directly through I-SceI cleavage. Sequence results were pooled from three independent sorting experiments for each cell line.

Figure 4.

Alternative end-joining mechanism is PARP1-dependent. (A) xrs5 cells were X-irradiated (20 Gy) and immunostained after 20 min for obtaining PAR. Pretreatment with the PARP-inhibitors (DIQ or NU1025) completely abrogated nuclear PAR accumulation. (B) Cell survival of CHOK1 and xrs5 cells after X-irradiation (closed symbols, solid lines) alone or in the presence of either PARP inhibitor (open symbols, dashed lines) was carried out. An amount of 75 µM DIQ (asterisk) and 300 µM NU1025 (diamonds) led to equal radiosensitization in xrs5 cells (dashed line), but had almost no effect on CHOK1 (open triangles). The mean values of three independent experiments are shown. Data were fitted to the linear quadratic equation S/S₀ = exp(–αD – βD²). The parameters obtained (α, β) were used to calculate the mean inactivation dose, D_bar (87) as a measure for the radiosensitivity. (C) NHEJ efficiency in the presence or absence of DIQ was determined as shown before (Figure 2) in CHOK1-pEJ and xrs5-pEJ clones (CHOK1-pEJ #70 and 71, and xrs5-pEJ #20 and 26). DIQ, given immediately after I-SceI transfection, significantly inhibited end-joining efficiency only in xrs5 cells. Data show the mean (±SE) of two experiments performed with two clones and three replicates each. (D) PARP1 was depleted from cells carrying pEJ by transient siRNA transfection 24 h before end-joining experiments were started as described before. (Immunoblot shows PARP1 expression 24 h after siRNA treatment.) The significant difference is indicated. sc indicates transfection with unspecific ‘scrambled’ siRNA. si indicates siRNA specifically targeting PARP1. Data show the mean (±SE) of two experiments performed with three replicates using a single clone of each strain.

Figure 5.

Complementing the Ku defect abrogates the PARP1-dependency of the end-joining process. Experiments were performed as described earlier. Xrs5-pEJ cells (clone #20) were co-transfected with I-SceI- and hKU80-expressing vectors (17) and analyzed for GFP⁺ after 24 h. Reversion of the NHEJ capability in xrs5 cells from three independent experiments was compared with the mean of CHOK1-pEJ taken from Figure 2A.

Figure 6.

PARP1-dependent end-joining is hindered by the presence of Ku but not modulated by DNA-PKcs. Cells were treated with either DIQ (75 µM) or the DNA-PK-inhibitor NU7026 (15 µM) or with both inhibitors simultaneously. (A) I-SceI was used to linearize pEJ plasmid in vitro of which 1.6 µg were then transfected using lipofectamine 2000 (Invitrogen, Germany) into DNA-PK-deficient (V3) and proficient (AA8) hamster cells. The % GFP⁺ cells were measured as an indicator of the repair after 24 and 48 h with or without DIQ. (B) Experiments were performed as described in Figure 4C. Inhibition of DNA-PKcs but not of PARP compromised the repair in CHOK1. (C) Inhibition of PARP but not of DNA-PK almost completely inhibited the slow repair in xrs5 cells.

Figure 7.

Model of Ku-dependent and independent DSB repair. In the presence of Ku, DSB repair is mainly executed by accurate NHEJ. A minor fraction is normally also repaired by HR or SSA (17). In the absence of Ku, repair is shuttled to all three other pathways which likely share a common initial end resection step. The lack of Ku binding leaves space for key proteins to bind such as PARP1, Rad51 or Rad52, respectively. The majority of DSBs are still repaired by end-joining, however by an alternative PARP1-dependent mode.