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 g-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.

Deficiency of the Arabidopsis Helicase RTEL1 Triggers a SOG1-Dependent Replication Checkpoint in Response to DNA Cross-Links

“…This is consistent with observations in C. elegans, in which rtel-1 is synthetically lethal with mus-81 (Barber et al, 2008). Since replicative stress triggers HR (Gao et al, 2012;Kalhorzadeh et al, 2014), the observed increased HR in rtel1-1 could at least partially result from replicative defects. Similar to the human RTEL1 helicase, the Arabidopsis RTEL1 may directly suppress HR by unwinding heteroduplex DNA (D-loop) (Barber et al, 2008).…”

“…We have speculated before that the HU-hypersensitive phenotype of WEE1-deficient plants arises because of their inability to coordinate the DNA replication rate with dNTP availability. This likely results in long stretches of single-stranded DNA that may become artificial substrates for homologous recombination, resulting in DNA deletions (Kalhorzadeh et al, 2014). It can be easily envisioned that the arrest of DNA helicases by DNA cross-links, arising through RTEL1 deficiency or MMC treatment, may counteract the unwinding of DNA and thus elevate the need for WEE1 (Figure 11).…”

The Structure-Specific Endonucleases MUS81 and SEND1 Are Essential for Telomere Stability in Arabidopsis

“…The absence of developmental or fertility defects in fancm send1 and recq4A send1 double mutant plants, neither of which showed reduced fertility (Supplemental Figure 5), suggests that SEND1 does not have a similar role in the resolution of toxic recombination intermediates, although a cryptic role in the absence of other nucleases remains possible. Another test involved RNaseH2 (Ribonuclease H2)-deficient plants, which show increased homologous recombination and in which the absence of MUS81 (mus81 trd1 [triffid 1] double mutant) causes a severe inhibition of root and shoot growth (Kalhorzadeh et al, 2014). However, like the trd1 mutant (Kalhorzadeh et al, 2014), send1 trd1 plants grow normally (Supplemental Figure 6).…”

“…Another test involved RNaseH2 (Ribonuclease H2)-deficient plants, which show increased homologous recombination and in which the absence of MUS81 (mus81 trd1 [triffid 1] double mutant) causes a severe inhibition of root and shoot growth (Kalhorzadeh et al, 2014). However, like the trd1 mutant (Kalhorzadeh et al, 2014), send1 trd1 plants grow normally (Supplemental Figure 6). Thus, while MUS81 plays an important role in HJ resolution in mitotic cells, SEND1 protein has at most a very minor role, and it is only in the absence of MUS81 that SEND1 becomes necessary.…”

“…For other mutants, we used the following alleles: mus81-2 (SALK_107515; Hartung et al, 2006), rad51-1 (GABI-KAT_134A01; Li et al, 2004), fancm-9 (SALK_120621; Crismani et al, 2012), recq4a-4 (Hartung et al, 2007, and RAD51-GFP (Da Ines et al, 2013). The RNaseH2 mutant, trd1-1 (Kalhorzadeh et al, 2014), was kindly provided by Lieven de Veylder.…”