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Deficiency of the Arabidopsis Helicase RTEL1 Triggers a SOG1-Dependent Replication Checkpoint in Response to DNA Cross-Links Zhubing Hu,a,b Toon Cools,a,b Pooneh Kalhorzadeh,a,b Jefri Heyman,a,b and Lieven De Veyldera,b,1 a Department b Department

of Plant Systems Biology, VIB, B-9052 Gent, Belgium of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Gent, Belgium

To maintain genome integrity, DNA replication is executed and regulated by a complex molecular network of numerous proteins, including helicases and cell cycle checkpoint regulators. Through a systematic screening for putative replication mutants, we identified an Arabidopsis thaliana homolog of human Regulator of Telomere Length 1 (RTEL1), which functions in DNA replication, DNA repair, and recombination. RTEL1 deficiency retards plant growth, a phenotype including a prolonged S-phase duration and decreased cell proliferation. Genetic analysis revealed that rtel1 mutant plants show activated cell cycle checkpoints, specific sensitivity to DNA cross-linking agents, and increased homologous recombination, but a lack of progressive shortening of telomeres, indicating that RTEL1 functions have only been partially conserved between mammals and plants. Surprisingly, RTEL1 deficiency induces tolerance to the deoxynucleotide-depleting drug hydroxyurea, which could be mimicked by DNA cross-linking agents. This resistance does not rely on the essential replication checkpoint regulator WEE1 but could be blocked by a mutation in the SOG1 transcription factor. Taken together, our data indicate that RTEL1 is required for DNA replication and that its deficiency activates a SOG1-dependent replication checkpoint.

INTRODUCTION Transmission of the genetic information in DNA across generations through replication requires the coordinated action of numerous multisubunit protein complexes, including the replisome and helicases (Knoll and Puchta, 2011; Bell and Kaguni, 2013; Leman and Noguchi, 2013; Popuri et al., 2013). Moreover, because DNA frequently suffers from spontaneous lesions induced by endogenous and exogenous factors, such as reactive oxygen species and DNA metabolic by-products, or stresses such as UV light, ionizing radiation, and DNA-damaging agents, cells have evolved a complex molecular machinery to sense and repair DNA lesions to maintain faithful DNA duplication (Yoshiyama et al., 2013, 2014). As part of this machinery, Ataxia Telangiectasia Mutated (ATM) and ATM- and RAD3-related (ATR) are two closely related kinases that play a central role in sensing and triggering DNA damage responses. ATM is primarily activated by DNA double-strand breaks (DSBs), whereas ATR responds to stalled replication forks and single-stranded DNA structures that interfere with DNA replication. In plants, as in other organisms, activated ATM or ATR transmits DNA damage signals to many downstream effectors, eventually arresting cell cycle progression and initiating DNA repair. Elements that arrest the cell cycle include the suppressor of gamma response 1 (SOG1) transcription factor, and the cell cycle inhibitory WEE1 kinase and SIAMESE-RELATED cyclin-dependent kinase inhibitors

1 Address

correspondence to [email protected] The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Lieven De Veylder (lieven. [email protected]). www.plantcell.org/cgi/doi/10.1105/tpc.114.134312

(SMR5 and SMR7) (De Schutter et al., 2007; Yoshiyama et al., 2009; Cools et al., 2011; Yi et al., 2014). Similar to ATR- and ATMdeficient plants, Arabidopsis thaliana plants with knockouts for these elements display normal vegetative development under standard growth conditions but exhibit growth defects in response to different kinds of DNA damage. In particular, knockout mutants of ATR or WEE1 are hypersensitive to replication-inhibitory drugs, demonstrating their importance for the repair of replication stressinduced DNA damage (Culligan, et al., 2004; De Schutter et al., 2007). By contrast, ATM and SOG1 are essential to react to DSBs, in part through the transcriptional activation of the SMR5 and SMR7 cell cycle inhibitory genes (Yi et al., 2014; Yoshiyama et al., 2014). Homologous recombination (HR) is critical for repairing DSBs and restarting stalled replication forks (Costes and Lambert, 2012). Furthermore, it is crucial for chromosomal pairing and exchange during meiosis (Humphryes and Hochwagen, 2014; Zamariola, et al., 2014). However, inappropriate HR can produce erroneous DNA rearrangements and intermediate recombination structures that cannot be resolved, resulting in genome instability (Krejci et al., 2012). Hence, HR must be tightly regulated and temporally coordinated with cell cycle progression and replication. In yeast and mammalian cells, several DNA helicases contribute to HR regulation by unwinding recombination intermediates or disrupting Rad51 nucleoprotein filaments (Colavito et al., 2010). The DNA helicase Regulator of Telomere Length 1 (RTEL1) is one of the proteins that suppresses HR through disassembling D-loop (two strands of a double-stranded DNA molecule separated for a stretch and held apart by a third strand of DNA) recombination intermediates (Barber et al., 2008). Caenorhabditis elegans rtel1 mutants exhibit elevated recombination rates and are synthetically lethal with deletion of the Bloom’s Syndrome helicase (BLM) homolog. This synthetic lethality correlates with

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the accumulation of recombination intermediates that persist and fail to be appropriately repaired. Deficiency of RTEL1 also results in an increased sensitivity to a range of DNA-damaging agents, in particular to DNA interstrand cross-links, which generate lesions affecting replication fork progression (Barber et al., 2008; Uringa et al., 2012). Apart from suppressing HR, mouse RTEL1 mediates DNA replication by its association with the proliferating cell nuclear antigen (PCNA) DNA clamp to avoid replication fork stalling or collapse (Vannier et al., 2013). Conversely, loss of the RTEL1PCNA interaction is accompanied by replication defects, such as reduced replication-fork extension rates, increased origin usage, and replication fork instability. In the aspect of telomeres, RTEL1 removes telomeric DNA secondary structures, T-loops, and telomeric G4-DNA to maintain telomere integrity, and in the absence of RTEL1, a rapid telomere shortening is observed (Vannier et al., 2012). Consistent with the pleiotropic functions of RTEL1, RTEL1 dysfunction in humans is associated with a range of cancers and with Hoyeraal-Hreidarsson syndrome, a rare X-linked recessive disorder (Deng et al., 2013; Vannier et al., 2014). Despite its importance in mammals, the functions of plant RTEL1 homologs remain to be examined. Here, we report the

characterization of Arabidopsis RTEL1. Analysis of T-DNA insertion mutants demonstrated that the plant RTEL1 plays a crucial role in DNA replication, repair, and recombination. Surprisingly, rtel1 mutants display an increased resistance to the replication inhibitory drug hydroxyurea (HU) that causes a depletion of deoxynucleotide triphosphates (dNTPs). This phenotype can be mimicked in wild-type plants by the administration of DNA interstrand cross-link-inducing agents and is independent of the replication checkpoint regulator WEE1, indicating that RTEL1 deficiency triggers a distinct replication checkpoint. RESULTS RTEL1KO Plants Exhibit Growth Inhibition Due to Cell Proliferation Defects Previous work led to the identification at least two types of DNA stress, replication defects and DSBs; these activate cell cycle checkpoints, resulting in a cell cycle delay and thus contributing to genome stability (Yoshiyama et al., 2013). To further dissect how plants maintain genome integrity, we performed

Figure 1. RTEL1-Deficient Plants Exhibit Growth Inhibition. (A) Morphology of 9-d-old wild-type (Col-0), rtel1-1, and rtel1-2 seedlings grown on half-strength MS medium. Bar = 1 mm. (B) to (D) Leaf growth of the first leaves of 3-week-old wild-type (Col-0), rtel1-1, and rtel1-2 plants. Leaf area (B), epidermal cell number (C), and epidermal cell size (D) on the abaxial side of the leaf. Data represent mean 6 SE (n = 5, **P value < 0.01, *P value < 0.05, two-sided Student’s t test). (E) Representative ploidy histograms of the first leaves of 3-week-old plants. (F) and (G) Roots of 7-d-old seedlings. Images of representative seedlings (F) and confocal microscopy images of plants stained with propidium iodide (G). Bars = 5 mm in (F) and 50 mm in (G). Arrowheads indicate the meristem size based on the cortical cell length.

RTEL1 Suppresses Replication Stress

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a systematic phenotypic analysis of genes that had been annotated as putative DNA replication/DNA repair regulators. One T-DNA insertion line (Salk_113285, hereafter referred to as rtel1-1) exhibited significant growth retardation of the young leaves and displayed a smaller final mature leaf size in comparison to wildtype plants (Figures 1A and 1B). Cellular analysis of mature leaves demonstrated that the decreased leaf area resulted from a reduction in cell number (Figure 1C), accompanied by a compensatory cell enlargement and increase in DNA endoreplication (Figures 1D and 1E; Supplemental Figure 1). Similarly, an inhibitory effect on root growth was observed, showing that rtel1-1 plants have a shorter primary root (Figure 1F). These phenotypes were confirmed by examination of an independent T-DNA insertion line (Salk_049464, rtel1-2) (Figure 1). In both lines, a reduction in root meristem size was observed (Figure 1G), correlated with a decrease in the number of cortical meristem cells (45.3 6 2.5 in wild-type plants versus 31.8 6 1.8 and 32.4 6 4.5 in rtel1-1 and rtel1-2, respectively [n > 5]). Similar to what we observed for the leaf cells, the reduction in dividing cells was accompanied by a compensatory increase in mature cell length (198.4 6 3.8 mm in wild-type plants versus 253.8 6 4.3 mm and 223.5 6 13.2 mm in rtel1-1 and rtel1-2, respectively [n > 100]). Thus, the observed root growth defect of the RTEL1 deficient plants is primarily due to an inhibition of cell division, rather than cell elongation. The T-DNA insertions in rtel1-1 and rtel1-2 are in the 7th exon and 16th intron of the RTEL1 gene (At1g77950), respectively (Supplemental Figure 2A). RT-PCR failed to detect full-length RTEL1 transcripts in the two mutants, suggesting that they are null (Supplemental Figure 2B). Through a BLAST search using the amino acid sequence of Arabidopsis RTEL1, we found 22

Figure 3. Depletion of RTEL1 Leads to Replication Defects.

Figure 2. Deficiency of Arabidopsis RTEL1 Does Not Cause Telomere Loss. (A) Telomere length comparison between wild type (Col-0) and rtel1 mutants of the 2nd generation. Genomic DNA was isolated from 8-d-old seedlings and digested with the restriction enzyme TruII. DNA gel blot analysis was performed using a digoxigenin-labeled telomere repeat as probe. (B) Morphology of 3-week-old wild type (Col-0), rtel1-1, stn1-1, and rtel1-1 stn1-1. Plants were isolated from an F2 segregating population generated by crossing rtel1-1 with stn1-1. Bars = 1 cm.

(A) Relative expression levels of DNA damage marker genes in wild-type (Col-0), rtel1-1, and rtel1-2 root tips. Expression levels in the wild type were arbitrarily set to one. All values were normalized against the expression level of the references genes. Data represent mean 6 SD (n = 3, **P value < 0.01, two-sided Student’s t test). (B) Quantification of the root length of 7-d-old wild-type (Col-0), rtel1-1, wee1-1, and rtel1-1 wee1-1 seedlings. (C) Quantification of the root length of 7-d-old wild-type (Col-0), rtel1-1, atr-2, and rtel1-1 atr-2 seedlings. Data represent mean 6 SD (n > 10, **P value < 0.01, *P value < 0.05, two-sided Student’s t test). (D) Representative confocal microscopy images of plants shown in (B) stained with propidium iodide. (E) Representative confocal microscopy images of plants shown in (C) stained with propidium iodide. Arrowheads indicate the meristem size based on the cortical cell length. Bars = 50 mm.

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Table 1. MMC Administration Exhibits a Positive Correlation with RTEL1 Deficiency at the Transcriptional Level Col-0 BRCA1 PARP2 RAD51 RNR1 SMR7 TSO2

1.00 1.00 1.00 1.00 1.00 1.00

6 6 6 6 6 6

sog1-1 0.006 0.057 0.030 0.034 0.029 0.063

0.95 0.81 0.94 0.92 0.95 0.97

6 6 6 6 6 6

MMC-Col-0 0.118 0.013 0.027 0.003 0.068 0.013

3.91 4.87 2.63 1.83 15.28 4.10

6 6 6 6 6 6

0.244 0.199 0.380 0.151 2.418 0.338

MMC-sog1-1

rtel1-1

6 6 6 6 6 6

5.15 4.55 2.88 1.81 15.76 3.23

1.83 2.44 1.31 1.38 4.58 1.39

0.124 0.171 0.032 0.102 1.210 0.046

6 6 6 6 6 6

rtel1-1 sog1-1 0.047 0.045 0.097 0.064 0.235 0.227

1.08 1.40 1.02 0.98 1.69 0.62

6 6 6 6 6 6

0.011 0.038 0.078 0.083 0.178 0.207

The 2- to 3-mm root tip of 7-d-old wild type (Col-0), sog1-1, rtel1-1, and rtel1-1 sog1-1 mutants grown on control medium or medium supplemented with 2.5 mg/L MMC were collected. Expression levels in Col-0 were arbitrarily set to one. All values were normalized against the expression level of the references genes. Data represent mean 6 SE (n = 2).

protein sequences with a high similarity to At-RTEL1 across six model species (Arabidopsis, Oryza sativa, Saccharomyces cerevisiae, C. elegans, Mus musculus, and Homo sapiens). Phylogenetic analysis showed that they classify into four groups, with At1g79950 falling into the RTEL1 group (Supplemental Figure 3A). Conserved domain analysis showed that Arabidopsis RTEL1 contains a Rad3-related DNA helicase (RAD3) domain, a PIP-box like domain, and a harmonin-N-like domain (Supplemental Figure 3B). Compared with Hs-RTEL1 and Mm-RTEL1, At-RTEL1 lacks one harmonin-N-like domain and a C4C4 domain or a RING-finger domain, indicating that the Arabidopsis orthologous gene might have lost a part of its gene functions (Supplemental Figure 3B). RTEL1 Deficiency Does Not Result in Telomere Shortening RTEL1 was originally characterized as a regulator of telomere length in mice, with embryonic stem cells deficient in RTEL1 displaying short telomeres (Ding et al., 2004). To test whether plant RTEL1 deficiency also causes telomere loss, we performed a terminal restriction fragments analysis on wild type, rtel1-1, and rtel1-2 mutants. Unexpectedly, all genotypes displayed identical telomere lengths (Figure 2A). Moreover, in contrast to known telomere mutants (Fitzgerald et al., 1999; Mozgová et al., 2010), no progressive shortening of the telomeres could be observed over generations, even in the 9th generation (Supplemental Figure 4A). STN1 is one component of the CST (CTC1, STN1, and TEN1) complex protecting chromosome ends in plants (Song, et al., 2008). To examine whether RTEL1 shows genetic interaction with the CST complex, we crossed rtel1-1 with stn1-1 and analyzed the F2 segregating population. As expected, both rtel1-1 and stn1-1 single mutants showed smaller leaves compared with wild-type plants (Figure 2B). The rtel1-1 stn1-1 double mutant exhibited deformed leaves (Figure 2B). Unexpectedly, the telomere length of stn1-1 rtel1-1 double mutants was not reduced but rather elongated compared with stn1-1 single mutants (Supplemental Figure 4B). Thus, the observed growth phenotypes observed for the rtel1 mutants are unlikely to originate because of telomere defects.

genes were differentially regulated (fold change $ 1.5; P value # 0.01), with 407 genes upregulated and 98 genes downregulated (Supplemental Data Set 1). Among a set of 61 genes identified before as DNA stress hallmark genes (Yi et al., 2014), 46 transcripts (75%) were upregulated in rtel1-1 mutant root meristems (Supplemental Table 1). In accordance, Gene Ontology (GO) analysis showed among the upregulated genes a significant overrepresentation of transcripts associated with response to ionizing radiation and DSB repair (Supplemental Figure 5). Transcriptional activation of DNA damage marker genes (such as PARP2, CYLINB1;1, and BRCA1) in rtel1 mutant seedlings was confirmed through a quantitative PCR approach (Figure 3A). These results indicated that RTEL1 deficiency generates damaged DNA. Based on the interaction of the mouse RTEL1 protein with PCNA (Vannier et al., 2013), we reasoned that the observed expression of DNA damage marker genes in rtel1-1 might be caused by problematic DNA replication. To confirm this hypothesis, we estimated the duration of the S-phase through measurement of the incorporation rate of ethynyl deoxyuridine (EdU), a thymidine analog

Depletion of RTEL1 Causes Replication Defects To understand the mechanism underlying the observed growth reduction of RTEL1 KO plants, an RNA sequencing experiment was conducted, comparing the transcriptome of rtel1-1 and Columbia-0 (Col-0) root meristems. In total, 505

Figure 4. RTEL1 Deficiency Triggers Increased HR. Recombination frequencies of wild-type (Control) and rtel1-1 seedlings using the 651 or IC9C reporters. Data represent mean number of GUS sectors 6 SD (n = 4, minimum 50 plants per repeat).

RTEL1 Suppresses Replication Stress

that can be incorporated into genomic DNA during replication (Hayashi et al., 2013). S-phase duration in rtel1-1 (4.21 h) was significantly prolonged in comparison with that in the wild type (2.01 h). To further emphasize that RTEL1-deficient plants suffer from problematic DNA replication, we introduced the rtel1-1 mutation into atr-2 and wee1-1 plants, which are hypersensitive to replication stress. Primary roots of rtel1-1 atr-2 and rtel1-1 wee1-1 double mutants were significantly shorter than those of rtel1-1 plants (Figures 3B and 3C) accompanied with shrinkage of their meristems (Figures 3D and 3E), indicating that both WEE1 and ATR suppress the rtel1 mutant phenotype. RTEL1 Suppresses HR As the HR marker gene RAD51 was highly induced in rtel1-1 mutants (Table 1; Supplemental Data Set 1), we postulated that RTEL1 deficiency might result in increased HR. To test this, we introduced two different HR substrates, 651 and IC9C, into the rtel1-1 background. Both substrate lines harbor two

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nonfunctional parts of the b-glucuronidase (GUS) gene (uidA) in a spatial orientation that can be restored into a functional uidA gene either by intra- and interchromosomal recombination in the 651 line or by only interchromosomal recombination in the IC9C line (Swoboda et al., 1994; Molinier et al., 2004; Schuermann et al., 2005). Both reporters revealed a significant increase in the level of spontaneous HR events in rtel1-1, compared with wild-type plants (Figure 4). The Arabidopsis MUS81 endonuclease is crucial for the resolution of Holliday junctions induced by HR (Hartung et al., 2006; Berchowitz et al., 2007). A synergistic effect on plant growth of rtel1-1 with mus81-1 was observed, since double mutants displayed a shorter root length and root meristem, compared with the mus81-1 single mutants that are phenotypically indistinguishable from wild-type plants (Figures 5A to 5C), confirming the increase in HR events in the rtel1 background. Similar to RTEL1, the Arabidopsis homolog of human BLM/ SGS1, i.e., RECQ4A, suppresses HR, since more HR events

Figure 5. RTEL1 Suppresses HR Independently from the RECQ4A Helicase. (A) Root growth of 7-d-old wild type (Col-0) and mus81-1, recq4a-4, rtel1-1, rtel1-1 mus81-1, and rtel1-1 recq4a-4 mutants. Bar = 5 mm. (B) Quantification of the root length of plants shown in (A). Data represent mean 6 SD (n > 10, **P value < 0.01, two-sided Student’s t test). (C) Representative confocal microscopy images of plants shown in (A) stained with propidium iodide. Bar = 50 µm. (D) Morphology of 6-week-old rtel1-1 and rtel1-1 recq4a-4 plants. Bar = 1 cm.

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Figure 6. RTEL1KO Mutants Are Hypersensitive to DNA Cross-Linking Agents but Tolerant to HU. Relative growth of primary roots of wild type (Col-0), rtel1-1, and rtel1-2 grown on the control medium and medium supplemented with DSB-producing agents 0.6 mg/L bleomycin (Bleo) or 5 mM zeocin (A), DNA cross-linking agents 2.5 mg/L mg CP or 2.5 mg/L MMC (B), or HU (C). Root length was measured 7 d after sowing. Data represent mean 6 SE (n > 10).

were observed in RECQ4A-deficient plants (Hartung et al., 2007). To define the genetic interaction between RECQ4A and RTEL1, we generated the rtel1-1 recq4a-4 double mutant. The recq4a-4 single mutant develops normally under nonstress conditions, albeit showing hypersensitivity to DNA

stresses (Hartung et al., 2007). In contrast, rtel1-1 recq4A-4 plants showed a disorganized root apical meristem and severely deformed shoots and roots, illustrating a synergistic interaction between RTEL1 and RECQ4A in plant growth (Figures 5A to 5D).

Figure 7. The HU Resistance Phenotype of the RTEL1KO Mutants Is Independent of Functional ATR and WEE1. (A) Root growth of 7-d-old wild type (Col-0), rtel1-1, atr-2, rtel1-1 atr-2, wee1-1, and rtel1-1 wee1-1 mutants grown on control medium (-HU, black bars) or medium supplemented with 0.5 mM HU (+HU, white bars). Data represent mean 6 SE (n > 10, **P value < 0.01, two-sided Student’s t test). (B) to (D) Representative confocal microscopy images of plants shown in (A) stained with propidium iodide. Arrowheads indicate the meristem size based on the cortical cell length. Bar = 50 mm.

RTEL1 Suppresses Replication Stress

RTEL1KO Mutants Are Hypersensitive to DNA Cross-Linking Agents Increased HR in rtel1 mutants suggested that RTEL1 deficiency may trigger DNA damage. To verify this, we determined the sensitivity of rtel1 mutants to genotoxic agents. Depletion of RTEL1 did not alter the sensitivity to bleomycin and zeocin, which trigger DSBs (Figure 6A). In contrast, rtel1 mutants were hypersensitive to mitomycin C (MMC) and cis-platin (CP), which trigger DNA crosslinking (Figure 6B). MMC mainly generates interstrand cross-links on DNA, whereas CP preferentially forms intrastrand cross-links. These results point out that RTEL1 has a function in the repair of cross-linked DNA. Based on the observed effect of RTEL1 deficiency on S-phase progression and suppression of the root growth phenotype by both ATR and WEE1 (Figures 3B to 3E), it is likely that cross-linked DNA may interfere with the progression of the replication fork. Replication fork inhibition can also be obtained by the depletion of dNTPs, which can be achieved through the supply of HU (Wang and Liu, 2006; Saban and Bujak, 2009). Intriguingly, rtel1 mutants were tolerant to HU, as observed by a decreased reduction in root growth, compared with control plants (Figure 6C). Cellular analysis illustrated that the HU tolerance phenotype of the rtel1-1 mutant plants resulted from maintenance

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of meristem cell number rather than an increase in cell length (Supplemental Figure 6). The HU tolerance induced by RTEL1 deficiency was even more pronounced in the WEE1- or ATR-deficient background (Figure 7A). Microscopy analysis confirmed that the observed growth differences were attributed to changes in meristem length (Figures 7B to 7D). Strikingly, whereas the meristems of the wee1-1 and atr-2 mutants were strongly affected by HU treatment due to the lack of the activation of a replication checkpoint, this meristem phenotype was strongly suppressed by the rtel1 mutation (Figures 7B to 7D), confirming that RTEL1 deficiency triggers a cell cycle checkpoint abrogating the need for ATR and WEE1. DNA Cross-Linking Agents Phenocopy the HU Resistance Phenotype Caused by RTEL1 Depletion Our aforementioned results suggested that the occurrence of cross-linked DNA might confer HU resistance. To test this hypothesis, we applied the DNA cross-linking drugs CP and MMC to wild-type plants. Similar to rtel1 mutants, both MMC-treated and CP-treated plants became tolerant to HU, as observed by a less pronounced reduction in root growth compared with that of control plants (Figures 8A and 8D) along with the absence of shrinkage of the root meristem (Figures 8B and 8E). Again, this phenotype

Figure 8. DNA Cross-Linkers Phenocopy the HU Resistance Phenotype of RTEL1KO Mutants. (A) Primary root length of wild type (Col-0), 2.5 mg/L MMC-treated Col-0 (MMC-Col-0), wee1-1, and 2.5 mg/L MMC-treated wee1-1 (MMC-wee1-1) treated without (-HU, black bars) or with 0.5 mM HU (+HU, white bars). (B) and (C) Representative confocal microscopy images of plants shown in (A) stained with propidium iodide. Bar = 50 mm. (D) Primary root length of wild type (Col-0), 1.25 mg/L CP-treated Col-0 (CP-Col-0), wee1-1, and 1.25 mg/L CP-treated wee1-1 (CP-wee1-1) treated without (-HU, black bars) or with 0.5 mM HU (+HU, white bars). Data represent mean 6 SE (n > 10, **P value < 0.01, two-sided Student’s t test). (E) and (F) Representative confocal microscopy images of plants shown in (D) stained with propidium iodide. Arrowheads indicate the meristem size based on the cortical cell length. Bar = 50 mm.

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was more pronounced for the HU-hypersensitive wee1-1 plants (Figures 8A and 8D), in which MMC or CP treatment rescued the strong effect of HU on meristem size (Figures 8C and 8F). These results support the idea that the type of damage induced by RTEL1 deficiency mimics that caused by cross-linked DNA, a hypothesis substantiated by quantitative PCR, demonstrating that genes being transcriptionally induced in rtel1 mutants show a similar activation as in wild-type plants treated with MMC (Table 1). The HU Resistance Phenotype Triggered by Cross-Linked DNA Depends on SOG1 Because the replication checkpoint induced by RTEL1 deficiency or DNA cross-linking agents appeared to be WEE1- and

Figure 10. SOG1 Controls a WEE1-Independent Replication Checkpoint.

Figure 9. The HU Resistance Phenotype Induced by Cross-Linked DNA Requires Functional SOG1, but Not ATM. (A) Root growth of 7-d-old atm-1, wee1-1, wee1-1 atm-1, sog1-1, and wee1-1 sog1-1 mutants grown on control medium (-HU), medium supplemented with 2.5 mg/L MMC, 0.5 mM HU, or 2.5 mg/L MMC + 0.5 mM HU (MMC+HU). (B) Root growth of 7-d-old wee1-1, rtel1-1 wee1-1, sog1-1, rtel1-1 sog1-1, wee1-1 sog1-1, and rtel1-1 wee1-1 sog1-1 mutants grown on control medium (-HU, black bars) or medium supplemented with 0.5 mM HU (+HU, white bars). Data represent mean 6 SE (n > 10, **P value < 0.01, two-sided Student’s t test).

(A) to (C) Root growth of 7-d-old wild type (Col-0), wee1-1, sog1-1, and wee1-1 sog1-1 mutants grown on control medium (-HU) or medium supplemented with 0.5 mM HU (+HU). Bar = 5 mm. (B) Quantification of root length of plants shown in (A). Data represent mean 6 SD (n > 10, **P value < 0.01, two-sided Student’s t test). (C) Representative confocal microscopy images of plants shown in (A) stained with propidium iodide. Arrowheads indicate the meristem size based on the cortical cell length. Bar = 50 mm.

RTEL1 Suppresses Replication Stress

ATR-independent (Figures 7 and 8), we tested whether the rescuing signaling pathway might depend on ATM and SOG1. To test this hypothesis, we generated atm-1 wee1-1 and sog1-1 wee1-1 double mutants because the MMC-conferring phenotype is clearer in the background of wee1-1 than that of wild-type plants. Plants were treated with MMC only or in combination with HU. Similar to wee1-1 single mutants, MMC administration to atm-1 single and atm-1 wee1-1 double mutants resulted in HU resistance, showing that the rescuing pathway does not rely on functional ATM (Figure 9A). By contrast, sog1-1 and sog1-1 wee1-1 mutants lost the HU resistance phenotype (Figure 9A). Similar to MMC administration, HU resistance induced by RTEL1 deficiency was also deprived in both sog1-1 and sog1-1 wee1-1 (Figure 9B). Additionally, quantitative PCR experiments confirmed that the expression of DNA stress genes induced by RTEL1 deficiency or MMC treatment depended predominantly on the SOG1 transcription factor (Table 1). The data obtained indicated that next to WEE1, SOG1 might be an essential replication checkpoint regulator. To confirm this hypothesis, we compared the sensitivity of wild-type, wee1-1, sog1-1, and wee1-1 sog1-1 plants to replication stress induced by HU. Similar to WEE1-deficient plants, sog1-1 was hypersensitive to HU, as observed by a reduction in root length and meristem size, compared with wild-type plants (Figure 10). Cellular visualization showed that sog1-1 root meristems exhibited a cell death phenotype under HU treatment, similar as that reported before for wee1-1 mutant plants (Figure 10C). Surprisingly, compared with the sog1-1 and wee1-1 single mutants, sog1-1 wee1-1 plants were extremely HU sensitive, with the sog1-1 wee1-1 double mutant root length being only 23.6 and 10.4% of wee1-1 and sog1-1 roots, respectively (Figure 10B). Moreover, in the sog1-1 wee1-1 double mutants, the meristem was totally consumed under replication stress conditions (Figure 10C). These data illustrate that SOG1 and WEE1 independently control the replication stress checkpoint.

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the protection of chromosome ends, both in plants and yeast (Puglisi et al., 2008; Song et al., 2008). In the absence of STN1, plants exhibit extensive loss of telomeric DNA, which triggers the ATM/ATR-mediated DNA damage response. We postulate that the effects of the mutation of rtel1-1 on DNA replication and HR might enhance the stringency of the activated cell cycle checkpoint pathways, with a synergistic growth inhibitory effect as a consequence. Simultaneously, as ATR contributes to telomere length maintenance (Amiard et al., 2011; Boltz et al., 2012), the rtel1-1 activated ATR pathway might account for the observed partial restoration of telomere length in rtel1-1 stn1-1 plants, compared with stn1-1. RTEL1-deficient plants showed a slow growth phenotype resulting from defective cell proliferation. This may be attributed to DNA replicative defects, triggering a cell cycle checkpoint to arrest DNA replication and cell cycle progression. In support of this, rtel1 mutants exhibited a prolonged S-phase, expression of DNA damage response genes, and synergetic growth defects with ATR- and WEE1-deficient plants, suggesting that Arabidopsis RTEL1 participates in DNA replication. In agreement, mouse RTEL1 associates with the replisome through binding to PCNA through its PIP box, which is conserved in the plant protein. Accordingly, disruption of the RTEL1-PCNA interaction compromises replication fork stability and slows down replication, inducing growth arrest and cell senescence (Vannier et al., 2013).

DISCUSSION In mammals and nematodes, RTEL1 controls different functions related to genome integrity (Ding et al., 2004; Vannier et al., 2014). Here, we show that deficiency of the Arabidopsis RTEL1 protein affects DNA replication, repair, and recombination, but we observed no obvious role in the control of telomere length. These data indicate that RTEL1’s role in DNA replication might be conserved but that the plant ortholog might have lost its role in telomere regulation. Correspondingly, plant RTEL1 proteins lack the C4C4 domain, whose mutation in the human RTEL1 results in elevated telomere loss (Le Guen et al., 2013; Vannier et al., 2014). Although phylogenetic analysis showed that Arabidopsis carries only one copy of the RTEL1 gene, supported by the phenotypic defects observed for the knockout plants, it cannot be excluded that RTEL1 might exhibit functional redundancy with its homologs in the regulation of telomere integrity. One particular candidate gene is FANCJ (At1g20750), whose expression is strongly induced in rtel1-1 mutants. Surprisingly, introducing the rtel1-1 mutation into an stn1-1 mutant background resulted in an increased telomere length, rather than the expected shortening. STN1 associates with TEN1 and CDC13 to form a trimeric complex that is critical for

Figure 11. Model for Replication Checkpoint Activation in Plants. HU treatment results in stalled replication forks that activate the WEE1 kinase in an ATR-dependent manner. By contrast, DNA cross-links induced by MMC treatment or absence of RTEL1 induce a SOG1-dependent inhibition of cell cycle progression, probably involving MAP kinases and the CDK inhibitor SMR proteins. Indirectly, the DNA cross-links may preactivate a replication checkpoint that confers HU resistance.

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The Plant Cell

In addition to replication defects, the absence of RTEL1 caused increased HR, supported by the phenotype of rtel1-1 mus81-1 double mutants. MUS81 is a highly conserved endonuclease and associates with the EME1 endonuclease to resolve HR intermediates, such as 39 flap structures and Holliday-like DNA junctions. The synergetic defects of rtel1-1 and mus81-1 indicate that the resolution of the HR intermediates arising from RTEL1 deficiency requires the MUS81/EME1 complex (Hartung et al., 2006; Mannuss et al., 2010). 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). The synthetic lethality of rtel1-1 with recq4a-4 illustrates that the HR repressing function of RTEL1 differs from that of SGS1/BLM, which prevents the formation of multichromatid joint molecules and dissolves double Holliday junctions in S. cerevisiae (Oh et al., 2007; Bernstein et al., 2010). Thus, plants appear to use different helicases to resolve distinct types of DNA structures that arise during replication, repair, and recombination. The increased sensitivity of rtel1 mutants to MMC and CP further supports RTEL1’s role in DNA replication and HR. CP and MMC induce intrastrand cross-links and interstrand crosslinks, respectively. The lack of sensitivity to bleomycin and zeocin, by which DSBs are generated, indicates that RTEL1 is specifically required for the repair events of DNA cross-links. Intriguingly, rtel1 mutant seedlings are more tolerant to HU compared with wild-type plants. Moreover, MMC and CP application mimic the HU resistance phenotype of rtel1-1 mutants, indicating that cross-linked DNA accounts for the observed HU resistance. HU is a direct inhibitor of ribonucleotide reductase (RNR) and its presence depletes the dNTP pool of cells, causing a slowdown of replication fork progression and a reduced activation of replication origins (Wang and Liu, 2006; Saban and Bujak, 2009). One mechanism to explain the observed HU resistance phenotype might be an upregulation of RNR expression, alleviating the dNTP drop. Alternatively, rtel1-induced DNA damage might preactivate a DNA damage response that confers HU resistance. DNA replication consists out of two steps: DNA unwinding by helicases and subsequent synthesis of the new DNA strands by DNA polymerases. The DNA replication checkpoint coordinates these two processes under the control of ATR. If dNTP levels are reduced, plants with a functional checkpoint will try to match their DNA helicase activity with the maximal polymerase activity given the available dNTP pool, e.g., by preventing the activation of new origins of replication. 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). As a result, long stretches of single-stranded DNA will not occur and only the repair of the cross-links is required.

Our genetic analysis indicates that this repair pathway depends on a functional SOG1, as the HU resistance phenotype was lost in rtel1 sog1-1 double mutant plants. Thus, next to its demonstrated role in sensing DSBs (Yoshiyama et al., 2009), SOG1 appears to control a replication-dependent checkpoint in response to DNA cross-links. The WEE1-dependent and SOG1-dependent pathways appear to be engaged upon the occurrence of stalled replication forks, based on the HU hypersensitive phenotype of the sog1-1 wee1-1 double mutants, in comparison to the sog1-1 and wee1-1 single mutants. Under these conditions, SOG1 is likely to be controlled by ATR (Yoshiyama et al., 2009). The cell division effectors operating downstream of SOG1 to cope with rtel1-induced DNA damage still await identification. Likely candidates include SMR4 and SMR7, representing two family members of the recently described class of SIAMESERELATED cyclin-dependent kinase inhibitors, of which at least SMR7 is under direct transcriptional control of SOG1 (Yi et al., 2014), since both genes show a strong transcriptional induction in RTEL1-deficient plants. Since DNA stress does not induce SOG1 transcription, SOG1 protein activity might be controlled at the posttranscriptional level. Three proteins from the mitogen-activated protein (MAP) kinase pathway, MAP kinase 3 (MPK3), MPK6, and MAP kinase phosphatase 1, were previously linked with DNA stress, namely, methyl methanesulfonate treatment and UV radiation, and operate independently from ATR (González Besteiro et al., 2011; González Besteiro and Ulm, 2013). Since the DNA cross-linking pathway is not controlled by ATR or ATM, it is an intriguing possibility that the MAP kinase pathway might control SOG1. Taken together, our results show that the absence of RTEL1 activity triggers DNA damage likely related to that induced by DNA cross-linking agents, illustrating an important role for the RTEL1 helicase in resolving aberrant replication structures. Moreover, next to its previously recognized role in DSB checkpoint control, our data identified the SOG1 transcription factor as an essential replication stress checkpoint regulator, working independently from WEE1. Our data illustrate that distinct types of DNA damages employ different signaling pathways to arrest DNA synthesis upon the occurrence of replication defects. METHODS Plant Materials and Growth Conditions Arabidopsis thaliana plants were grown under long-day conditions (16 h of light/8 h of darkness) at 22°C on half-strength Murashige and Skoog (MS) germination medium (Murashige and Skoog, 1962). The rtel1-1 (SALK_113285) and rtel1-2 (SALK_046494) alleles were acquired from the ABRC. Homozygous insertion alleles were checked by genotyping PCR using the primers listed in Supplemental Table 4. The atm-1, atr-2, wee1-1, sog1-1, mus81-1, and recq4a-4 mutants have been described previously (Garcia et al., 2003; Culligan et al., 2004; De Schutter et al., 2007; Yoshiyama et al., 2009; Hartung et al., 2006, 2007; Chen et al., 2008). For the treatments with genotoxic agents, seeds were directly plated on control medium and medium supplemented with the indicated drugs. The root length of 7-d-old seedlings was measured. Microscopy and Flow Cytometric Analyses For leaf measurements, the first leaves of 21-d-old plants were cleared overnight in ethanol and stored in lactic acid. The leaf area, the cell area of

RTEL1 Suppresses Replication Stress

pavement cells, and the total number of cells per leaf were obtained as described previously (Yi et al., 2014). For root meristem observations, root tips of 7-d-old seedlings were stained for 3 min in a 10 mM propidium iodide solution (Sigma-Aldrich), washed for 15 min with Milli-Q water, and visualized with an LSM 5 exciter confocal microscope (Zeiss). For flow cytometric analysis, leaf material was chopped in 200 mL of Cystain UV Precise P nuclei extraction buffer (Partec), supplemented with 800 mL of staining buffer. The filtered supernatants were measured by the Cyflow MB flow cytometer (Partec). The nuclei were analyzed with the CyFlow flow cytometer using FloMax (Partec) software. Phylogenetic Tree Construction and Conserved Domains Analysis RTEL1 homologs were identified from GenBank using the protein basic local alignment search tool (BLASTp) (http://blast.ncbi.nlm.nih.gov/Blast. cgi). The alignment of full-length amino acid sequences was used to construct the neighbor-joining tree using the MEGA3 (Molecular Evolutionary Genetic Analyses, version1.1, Pennsylvania State University; http:// www.megasoftware.net/) package. Conserved domains of Arabidopsis RTEL1 were predicted based on the protein structure of human and mouse RTEL1 homologs described by Vannier et al. (2014).

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S-Phase Duration Evaluation Four-day-old seedlings grown vertically on half-strength MS were transferred to liquid medium (0.53 MS, 1% sucrose, and 10 mM EdU in Click-iT component A [Invitrogen]) incubated with EdU at 22°C under long-day conditions (16 h of light/8 h of darkness). Sample collection, EdU detection, and S-phase evaluation were performed as described previously (Hayashi et al., 2013). Accession Numbers RNA sequencing data have been submitted to ArrayExpress (www.ebi.ac. uk/arrayexpress) under accession number E-MTAB-2943. Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: ATM (At3g48190), ATR (At5g40820), MUS81 (At4g30870), RECQ4A (At1g10930), RTEL1 (At1g79950), STN1 (At1g07130), SOG1 (At1g25580), and WEE1 (At1g02970). Supplemental Data Supplemental Figure 1. DNA Ploidy Level Distribution of Col-0, rtel1-1, and rtel1-2. Supplemental Figure 2. Isolation of rtel1 Mutants.

RNA Sequencing and GO Analysis Supplemental Figure 3. Phylogenetic Analysis of RTEL1. Root tips (

Deficiency of the Arabidopsis helicase RTEL1 triggers a SOG1-dependent replication checkpoint in response to DNA cross-links.

To maintain genome integrity, DNA replication is executed and regulated by a complex molecular network of numerous proteins, including helicases and c...
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