Arabidopsis thaliana RNase H2 deficiency counteracts the needs for the WEE1 checkpoint kinase but triggers genome instability

Abstract

The WEE1 kinase is an essential cell cycle checkpoint regulator in Arabidopsis thaliana plants experiencing replication defects. Whereas under non-stress conditions WEE1-deficient plants develop normally, they fail to adapt to replication inhibitory conditions, resulting in the accumulation of DNA damage and loss of cell division competence. We identified mutant alleles of the genes encoding subunits of the ribonuclease H2 (RNase H2) complex, known for its role in removing ribonucleotides from DNA-RNA duplexes, as suppressor mutants of WEE1 knockout plants. RNase H2 deficiency triggered an increase in homologous recombination (HR), correlated with the accumulation of γ-H2AX foci. However, as HR negatively impacts the growth of WEE1-deficient plants under replication stress, it cannot account for the rescue of the replication defects of the WEE1 knockout plants. Rather, the observed increase in ribonucleotide incorporation in DNA indicates that the substitution of deoxynucleotide with ribonucleotide abolishes the need for WEE1 under replication stress. Strikingly, increased ribonucleotide incorporation in DNA correlated with the occurrence of small base pair deletions, identifying the RNase H2 complex as an important suppressor of genome instability.

Figure 1.

A Mutation in the Catalytic Subunit of the RNase H2 Complex Partially Rescues HU Hypersensitivity of WEE1^KO Plants. (A) and (B) Root growth of 7-d-old wild-type (Col-0) and wee1-1, trd1-1 wee1-1, and trd1-2 wee1-1 mutant plants grown on control medium (A) or medium supplemented with 0.75 mM HU (B). Bar = 0.5 cm. (C) Quantification of the root length of plants shown in (A) and (B). Data represent mean ± sd (n > 10, **P value < 0.01, two-sided Student’s t test). (D) and (E) Representative confocal microscopy images of plants shown in (A) and (B) stained with propidium iodide. Arrowheads indicate the meristem size based on the cortical cell length. Bar = 50 μm. (F) Number of meristematic cortex cells. Data represent mean ± sd (n > 10, **P value < 0.01, two-sided Student’s t test). [See online article for color version of this figure.]

Figure 2.

TRD1 Encodes the Catalytic Subunit of the RNase H2 Complex. (A) Intron-exon organization of TRD1. Black and gray boxes represent exons and untranslated regions, respectively. The position of the mutated base pair (trd1-1) and T-DNA insertion site (trd1-2) are indicated. (B) Sequence alignment of the RNase H2 subunit A from different species highlighting the conserved position of the Gly residue (indicated by a star). Mm, Mus musculus; Hs, Homo sapiens; Os, Oryza sativa; Zm, Zea mays; At, Arabidopsis thaliana; Sc, Saccharomyces cerevisiae.

Figure 3.

The Arabidopsis RNase H2 Complex Comprises Three Subunits. (A) and (B) Protein-protein interaction between different RNase H2 subunits as identified by tandem affinity purification from cell cultures cultivated in the absence (A) or presence of 10 mM HU for 24 h (B). Arrows point from bait to prey and correspond to interactions that were confirmed in a repeat experiment (Supplemental Data Set 2). (C) Yeast two-hybrid interactions between the different subunits of RNase H2. The GUS protein was used as negative control.

Figure 4.

RNase H2 Deficiency Triggers Increased HR. Recombination frequencies of untreated (black bars) and HU-treated (0.75 mM; gray bars) control (Col-0), trd1-2, trd1-2 wee1-1, and wee1-1 seedlings using the 651 (A) or IC9C (B) reporters. Data represent mean number of GUS sectors ± se (n = 4, minimum 50 plants per repeat).

Figure 5.

Lack of RNase H2 Activity Triggers the Accumulation of γ-H2AX Foci. (A) Average number of γ-H2AX foci per nucleus of wild-type (Col-0) and wee1-1 mutants, untreated or treated with HU. Data represent mean ± sd (n = 100, *P value < 0.05, two-sided Student’s t test). (B) Detection of γ-H2AX foci in wild-type (Col-0), wee1-1, trd1-2, and wee1-1 trd1-2 root tip cells untreated (−HU) or treated with 1 mM HU (+HU). Bar = 2 μm. (C) γ-H2AX immunofluorescence in S- or early G2-phase nuclei of trd1-2 mutants (being Edu positive). Bar = 2 μm.

Figure 6.

Mutations in XRCC2 and RAD51C Partially Rescue WEE1^KO HU Hypersensitivity. (A) and (B) Root growth of 7-d-old wild-type (Col-0) and xrcc2-1, wee1-1, and xrcc2-1 wee1-1 mutant plants grown on control medium (A) or medium supplemented with 0.75 mM HU (B). Bar = 0.5 cm. (C) Quantification of the root length of plants shown in (A) and (B). Data represent mean ± sd (n > 10, **P value < 0.01, two-sided Student’s t test). (D) and (E) Root growth of 7-d-old wild-type (Col-0) and rad51c-1, wee1-1, and rad51c-1 wee1-1 mutant plants grown on control medium (D) or medium supplemented with 0.75 mM HU (E). Bar = 0.5 cm. (F) Quantification of the root length of plants shown in (D) and (E). Data represent mean ± sd (n > 10, **P value < 0.01, two-sided Student’s t test). [See online article for color version of this figure.]

Figure 7.

RNase H2-Deficient Plants Accumulate rNMPs in DNA. (A) Alkaline cleavage products of genomic DNA extracted from 7-d-old wild-type (Col-0), wee1-1, trd1-2, and trd1-2 wee1-1 seedlings grown under control conditions or in the presence of 0.75 mM HU. (B) Densitometry plot of lanes in (A).

Figure 8.

Lack of RNase H2 Activity Causes Small Base Pair Deletions. Sequencing reads of mutant loci in first versus third generation plants. Deleted base pairs (indicated by dots) result in dual sequence reads.