Function of the Plant DNA Polymerase Epsilon in Replicative Stress Sensing, a Genetic Analysis

Abstract

Faithful transmission of the genetic information is essential in all living organisms. DNA replication is therefore a critical step of cell proliferation, because of the potential occurrence of replication errors or DNA damage when progression of a replication fork is hampered causing replicative stress. Like other types of DNA damage, replicative stress activates the DNA damage response, a signaling cascade allowing cell cycle arrest and repair of lesions. The replicative DNA polymerase ε (Pol ε) was shown to activate the S-phase checkpoint in yeast in response to replicative stress, but whether this mechanism functions in multicellular eukaryotes remains unclear. Here, we explored the genetic interaction between Pol ε and the main elements of the DNA damage response in Arabidopsis (Arabidopsis thaliana). We found that mutations affecting the polymerase domain of Pol ε trigger ATR-dependent signaling leading to SOG1 activation, WEE1-dependent cell cycle inhibition, and tolerance to replicative stress induced by hydroxyurea, but result in enhanced sensitivity to a wide range of DNA damaging agents. Using knock-down lines, we also provide evidence for the direct role of Pol ε in replicative stress sensing. Together, our results demonstrate that the role of Pol ε in replicative stress sensing is conserved in plants, and provide, to our knowledge, the first genetic dissection of the downstream signaling events in a multicellular eukaryote.

Figure 1.

The abo4-1 mutant shows increased tolerance to HU-induced replicative stress. A and B, Wild-type (Col-0) and abo4-1 mutant seedlings were germinated on HU-supplemented medium and plants with true leaves were counted after 12 d. The atr mutant was used as a hypersensitive control. In , values are average ± se of three biological replicates. Asterisks indicate statistically relevant differences with respect to the wild type in the same conditions (Student t test, P < 0.05). C to E, Wild type (Col-0) and abo4-1 mutant seedlings were grown for 4 d on half-strength MS and transferred to HU-supplemented medium (1 mM) for 9 d to monitor root growth. C, By contrast with wild-type plants, root length was almost unchanged by HU exposure in the abo4-1 mutant; arrowheads mark the position of the root tip. D, Average root length was measured after 9 d on HU; at least 20 plantlets were used for each treatment; values are average ± se. Different letters indicate significantly different values (Student t test, P < 0.05). Data are representative of two independent experiments. E, The relative growth of each genotype after 9 d on HU was calculated compared to untreated plants of the same genotype.

Figure 2.

ATR and WEE1 are required for abo4-1 mutant viability. A to F, Open siliques of wild type (A), atr (B), wee1 (C), abo4-1 (D) mutants, and atr/+ abo4-1 (E) and wee1/+ abo4-1 (F) sesquimutants. Arrows point to aborted seeds. Bar = 2 mm for all panels. G to N, Embryo development in wild type (G to J) and wee1/+ abo4-1 sesquimutants. G, Globular stage. H, Late heart stage. I and J, Early and late torpedo stage. In the siliques of wee1/+ abo4-1 mutants, approximately 3/4 of embryos undergo normal development as in the wild type, abo4-1, or wee1 single mutants. However, 1/4 of embryos stop development at various stages and show aberrant division patterning. K, Arrested embryo just after fertilization; the arrow points to the single nucleus of the endosperm. L and M, Embryos at the globular stage with abnormal cell organization. N, Embryo at the late torpedo stage with misshapen cotyledons. Bar = 50 µm for all panels.

Figure 3.

The checkpoint activated by the abo4-1 mutation is partially dependent on SOG1. A and B, HU sensitivity in wild type (Col-0), abo4-1, sog1, and abo4-1 sog1 mutants. Plantlets were grown on half-strength MS for 4 d and transferred to control medium or HU-supplemented medium for 9 d. A, Root length; values are average ± se obtained on at least 15 plantlets. B, Relative root growth; values are expressed as percentage of length on MS medium. C, qRT-PCR analysis of the expression of selected genes in abo4-1, sog1, and abo4-1 sog1 mutants; values are average ± sd. D and E, Zeocin sensitivity in wild-type (Col-0), abo4-1, sog1, and abo4-1 sog1 mutants. Plantlets were grown on half-strength MS for 4 d and transferred to control medium (full bars) or zeocin-supplemented medium (10 µM, dashed bars) for 9 d. D, Root length; values are average ± se obtained on at least 15 plantlets. In A and D, different letters indicate significantly different values (Student t test, P < 0.05). For all panels, data are representative of at least two independent experiments.

Figure 4.

The checkpoint activated by the abo4-1 mutation is ATM-independent. A and B, HU sensitivity in wild type (Col-0), abo4-1, atm, and abo4-1 atm mutants. Plantlets were grown on half-strength MS for 4 d and transferred to control medium (full bars) or HU-supplemented medium (1 mM, dashed bars) for 9 d. A, Root length; values are average ± se obtained on at least 15 plantlets. B, Relative root growth; values are expressed as percentage of length on MS medium. C, qRT-PCR analysis of the expression of selected genes in abo4-1, atm, and abo4-1 atm mutants; values are average relative expression compared to the wild type ± sd. D, Zeocin sensitivity in wild type (Col-0), abo4-1, atm, and abo4-1 atm mutants. Plantlets were grown on half-strength MS for 4 d and transferred to control medium (full bars) or zeocin-supplemented medium (10 µM, dashed bars) for 9 d. D, Root length; values are average ± se obtained on at least 15 plantlets. E, Representative picture of plantlets grown on half-strength medium (mock), or grown on MS supplemented with zeocin (10 µM) for 6 d and allowed to recover for another 6 d. Arrowheads mark the position of the root tip. F, Relative fresh weight of plantlets after recovery. Values are average ± se from six replicates. In A, D, and F, different letters indicate statistically relevant differences (Student t test, P < 0.05).

Figure 5.

Proper levels of POL2A is required for checkpoint activation in DDR. A and B, Wild-type (Col-0) and POL2A-RNAi seedlings were grown for 4 d on half-strength MS and transferred to HU-supplemented medium (1 mM) for 9 d. POL2A-RNAi lines were hypersensitive to this drug: lines indicate the extremity of roots (A). After 9 d, root length was measured on plants kept on control medium (full bars) or on HU-supplemented medium (dashed bars; B). Values above the bar indicate the relative root growth compared to the respective untreated control. C and D, Wild-type (Col-0) and POL2A-RNAi seedlings were grown for 4 d on half-strength MS and transferred to zeocin-supplemented medium (10 µM) for 9 d. POL2-RNAi lines were unaffected by this drug (C). Arrowheads mark the position of the root tip. After 9 d, root length was measured on plants kept on control medium (full bars) or on zeocin-supplemented medium (dashed bars; D). E, qRT-PCR quantification of selected genes in POL2A-RNAi seedlings. Values are average ± sd compared to the wild type. F, POL2A-RNAi plantlets are hypersensitive to IATM (Ku55933). Plants were germinated on MS medium containing DMSO or IATM (10 µM). After 10 d, the percentage of plants with true leaves was monitored. Germination and development are severely affected in POL2A-RNAi lines, and the proportion of plants with true leaves was therefore reduced compared to the wild type on control medium. However, this reduction was even more pronounced in the presence of IATM, whereas this compound had no effect on wild-type plants. In B, D, and E, values are average ± se of data obtained on at least 15 plantlets. Different letters indicate statistically relevant differences (Student t test, P < 0.05). All data are representative of at least two biological replicates.

Figure 6.

abo4-2 mutants show SOG1-dependent meiotic fragmentation. Meiosis progression in the wild type (A to E), abo4-2 mutant (F to I), and abo4-2 sog1 mutant. In the wild type, after early prophase (A), bivalents were formed (B), homologous chromosomes segregated during division I, and sister chromatids segregate during division II (C, metaphase; D, anaphase) to form tetrads (E). In the abo4-2 mutant, early prophase was normal (F), but bivalents were never observed. Instead, in 90% of the cells, extensive DNA fragmentation was observed both during the first (G) and the second division (H), leading to the formation of polyads (I). In abo4-2 sog1 mutants, 40% of the cells still showed DNA fragmentation (J), but 60% of meiocytes were wild-type-like (K, end of division I; L, anaphase of division II; M, tetrad). Bar = 10 µm for all panels.

Figure 7.

Model for Pol ε function in plant DDR. A, In the wild type, Pol ε catalytic subunit POL2A is involved in replication stress sensing; this leads to ATR-dependent activation of the WEE1 and SOG1 pathways, allowing the expression of genes involved in cell cycle arrest, DNA repair, and nucleotide biosynthesis, ultimately leading to fork stabilization and completion of DNA replication and cell survival. B, In pol2A mutants with point mutations affecting POL2A activity, the abnormal Pol ε subunit likely gums up replication, leading to constitutive replication stress and activating ATR. The WEE1 branch of the downstream pathway is essential to plant survival, whereas the SOG1 branch of the pathway is dispensable, but confers tolerance to replicative stress. SOG1 activation may also negatively regulate ATM signaling leading to enhanced sensitivity to DNA damaging agents. C, When accumulation of POL2A is reduced, Pol δ likely replaces it and replicates both DNA strands. In the absence of Pol ε, replicative stress signaling is not properly activated, which may lead to fork collapse and DNA lesions that can in turn activate ATM signaling and promote tolerance to DSB-inducing agents. In all panels, dashed arrows indicate putative pathways that remain to be molecularly identified.