Abrogation of the CLK-2 checkpoint leads to tolerance to base-excision repair intermediates

Abstract

Incorporation of uracil during DNA synthesis is among the most common types of endogenously generated DNA damage. Depletion of Caenorhabditis elegans dUTPase by RNA interference allowed us to study the role of DNA damage response (DDR) pathways when responding to high levels of uracil in DNA. dUTPase depletion compromised development, caused embryonic lethality and led to activation of cell-cycle arrest and apoptosis. These phenotypes manifested as a result of processing misincorporated uracil by the uracil-DNA glycosylase UNG-1. Strikingly, abrogation of the clk-2 checkpoint gene rescued lethality and developmental defects, and eliminated cell-cycle arrest and apoptosis after dUTPase depletion. These data show a genetic interaction between UNG-1 and activation of the CLK-2 DDR pathway after uracil incorporation into DNA. Our results indicate that persistent repair intermediates and/or single-stranded DNA formed during repair of misincorporated uracil are tolerated in the absence of the CLK-2 checkpoint in C. elegans.

Figure 1

The de novo pathway for thymidine biosynthesis. A simplified scheme is shown. Use of dUTP for DNA synthesis in place of dTTP can be promoted by depletion of dUTPase.

Figure 2

dut-1(RNAi) induces cell-cycle arrest and apoptosis in wild-type animals. (A) The presence of fewer but enlarged 4,6-diamidino-2-phenylindole-stained nuclei in the mitotic region of dissected gonads shows induction of cell-cycle arrest in dut-1(RNAi)-fed N2 animals. Germ lines extruded from untreated (−) and γ-irradiated (75 Gy γ-IR) animals are shown for comparison. The number of nuclei was counted in a volume of 54,000 μm³ 16 h after the exposure of L4 larvae to the indicated treatments in 25 germ lines. Scale bars, 5 μm. (B) Induction of apoptosis was scored by counting apoptotic corpses in the pachytene region of the germ line under differential interference contrast before (−) and after (+) ionizing radiation (IR), and after dut-1(RNAi). Error bars indicate the standard error of the mean for at least 20 adult worms from three independent experiments. RNAi, RNA interference; Wt, wild type.

Figure 3

Rescue of dut-1(RNAi) in ung-1 and clk-2 mutants. (A) Differential interference contrast images of representative phenotypes observed in the progeny in empty vector L4440(RNAi) control-fed (a–c) compared with dut-1(RNAi)-fed (d–f) animals. N2 worms fed dut-1(RNAi) develop a strong protruding vulva (Pvl) phenotype (d), which was fully rescued in the clk-2 mutant (e) but only partly rescued in ung-1, as some animals showed a Pvl phenotype (f, arrow pointing to Pvl in the inset). Scale bars, 10 μm. (B) Cell-cycle arrest and (C) apoptotic corpses per germ line were counted 16 h after exposing L4 to dut-1(RNAi) (open bars) compared with animals fed empty vector control, L4440(RNAi) (filled bars). Error bars indicate the standard error of the mean for at least 20 adult worms from three independent experiments. RNAi, RNA interference; Wt, wild type.

Figure 4

Recruitment of ATL-1 and RAD-51 to chromatin after dut-1(RNAi) depends on UNG-1. Representative images of anti-ATL-1 (top left, A) and anti-RAD-51 (top right, C) staining of fixed mitotic nuclei from N2 wild-type and clk-2 mutant animals 16 h after exposure of L4 larvae to the indicated RNAi. (B,D) Quantification of the data is shown for N2 (filled) and clk-2 (open) bars. Error bars indicate the standard error of the mean for at least 20 adult worms from two independent experiments. RNAi, RNA interference; Wt, wild type.