Mutation in DDM1 inhibits the homology directed repair of double strand breaks

Abstract

In all organisms, DNA damage must be repaired quickly and properly, as it can be lethal for cells. Because eukaryotic DNA is packaged into nucleosomes, the structural units of chromatin, chromatin modification is necessary during DNA damage repair and is achieved by histone modification and chromatin remodeling. Chromatin remodeling proteins therefore play important roles in the DNA damage response (DDR) by modifying the accessibility of DNA damage sites. Here, we show that mutation in a SWI2/SNF2 chromatin remodeling protein (DDM1) causes hypersensitivity in the DNA damage response via defects in single-strand annealing (SSA) repair of double-strand breaks (DSBs) as well as in the initial steps of homologous recombination (HR) repair. ddm1 mutants such as ddm1-1 and ddm1-2 exhibited increased root cell death and higher DSB frequency compared to the wild type after gamma irradiation. Although the DDM1 mutation did not affect the expression of most DDR genes, it did cause substantial decrease in the frequency of SSA as well as partial inhibition in the γ-H2AX and Rad51 induction, the initial steps of HR. Furthermore, global chromatin structure seemed to be affected by DDM1 mutations. These results suggest that DDM1 is involved in the homology directed repair such as SSA and HR, probably by modifying chromatin structure.

Fig 1. ddm1 mutants show hypersensitive phenotypes after gamma irradiation.

(A) Physical map of DDM1 and its mutations in ddm1 mutants. (B) Phenotypes of Col-0 (WT) and ddm1 mutants after exposure to gamma radiation. Images were taken 14 days after gamma irradiation. CT, control; GR, gamma radiation. Digits in parenthesis indicate a dose of gamma radiation.

Fig 2. DDM1 mutation increases cellular DNA damage upon exposure to gamma radiation.

(A) Representative images of root tips, which were stained with propidium iodide 1 day after gamma irradiation. Scale bars: 50 μm. (B) Diagram to show extent of dead cell area. (C) Proportion of dead cell area in root meristem. Bars represent means ± SE (n = 19) from three independent experiments. (D) Representative comet images of nuclei and (E) proportion of nuclei with more than 40% tail DNA in WT and ddm1 mutants without or with exposure to 200 Gy of gamma radiation. Bars represent means ± SE (n = 3) of three independent experiments using 250 nuclei. Different letters in (C) and (E) indicate significant differences among the samples at a threshold of P < 0.05 [one-way ANOVA, Tukey’s honestly significant difference (HSD) test]. CT, control; GR, gamma radiation.

Fig 3. Radiation-sensitive phenotypes of ddm1 mutants are not due to the altered transcription of DDR genes.

Quantitative real-time PCR analysis of the expression of DDR genes in WT and ddm1 mutants 2 and 24 h after gamma irradiation. BRCA1, breast cancer 1; RPA1E, replication protein A 1e; PARP1, poly [ADP-ribose] polymerase 1; RAD51, radiation sensitive 51. Bars represent means ± SE (n = 3) of three independent experiments. ACTIN2 was used as an endogenous control gene.

Fig 4. DDM1 mutation causes defects in SSA repair of DSBs after gamma irradiation.

(A) Physical map of the DGU.US-1 construct. (B) Model of SSA pathway for repair of DSBs (modified from [8, 9]). (C) Images of a whole seedling and a leaf showing blue spots, which represent SSA events in the reporter DGU.US line. Scale bars: 5 and 0.5 mm, respectively. (D) Change in the frequency of SSA events in DGU.US and ddm1/DGU.US lines after gamma irradiation. Bars represent means ± SE (n = 120) of three independent experiments. Different letters indicate significant differences among the samples at a threshold of P < 0.05 (one-way ANOVA, Tukey’s HSD test). CT, control; GR, gamma radiation.

Fig 5. ddm1 mutants have defects in induction of γ-H2AX and RAD51.

(A) Immunoblot of γ-H2AX and (B) RAD51 in WT and ddm1 mutants after gamma irradiation. CT, control; GR, gamma radiation.

Fig 6. DDM1 mutation affects chromatin structure before and after gamma irradiation.

(A) Chromatin digestion with micrococcal nuclease (MNase). (B) Proportion of MNase-digested chromatin DNA resolved on agarose gels. The genomic band intensities without (g₀) and with (g_c) treatment with different concentrations of MNase were quantified, and the ratio of g_c/g₀ was used to represent the degree of chromatin relaxation. Bars represent means ± SE (n = 3) of three independent experiments. CT, control; GR, gamma radiation.

Fig 7. Complementation of DDM1 mutation by overexpressing the full-length cDNA of DDM1 in the ddm1 mutant is effective.

(A) Phenotypes of transgenic lines overexpressing the DDM1 cDNA in WT and ddm1 mutant backgrounds in response to gamma irradiation. Seedlings were grown for 18 days after gamma irradiation at 200 Gy for 4 h. CT, control; GR, gamma radiation. (B) Immunoblot of γ-H2AX in transgenic lines overexpressing the DDM1 cDNA in WT and ddm1 mutant backgrounds after gamma irradiation at 200 Gy.