Deficiency of the Arabidopsis Helicase RTEL1 Triggersa SOG1-Dependent Replication Checkpoint in Response toDNA 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 of Plant Systems Biology, VIB, B-9052 Gent, BelgiumbDepartment 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, weidentified 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 anddecreased cell proliferation. Genetic analysis revealed that rtel1 mutant plants show activated cell cycle checkpoints, specificsensitivity to DNA cross-linking agents, and increased homologous recombination, but a lack of progressive shortening oftelomeres, indicating that RTEL1 functions have only been partially conserved between mammals and plants. Surprisingly, RTEL1deficiency induces tolerance to the deoxynucleotide-depleting drug hydroxyurea, which could be mimicked by DNA cross-linkingagents. This resistance does not rely on the essential replication checkpoint regulator WEE1 but could be blocked by a mutationin the SOG1 transcription factor. Taken together, our data indicate that RTEL1 is required for DNA replication and that its deficiencyactivates a SOG1-dependent replication checkpoint.

INTRODUCTION

Transmission of the genetic information in DNA across gen-erations through replication requires the coordinated action ofnumerous multisubunit protein complexes, including the repli-some 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 in-duced by endogenous and exogenous factors, such as reactiveoxygen species and DNA metabolic by-products, or stressessuch as UV light, ionizing radiation, and DNA-damaging agents,cells have evolved a complex molecular machinery to senseand repair DNA lesions to maintain faithful DNA duplication(Yoshiyama et al., 2013, 2014). As part of this machinery, AtaxiaTelangiectasia Mutated (ATM) and ATM- and RAD3-related(ATR) are two closely related kinases that play a central role insensing and triggering DNA damage responses. ATM is primarilyactivated by DNA double-strand breaks (DSBs), whereas ATRresponds to stalled replication forks and single-stranded DNAstructures that interfere with DNA replication. In plants, as inother organisms, activated ATM or ATR transmits DNA damagesignals to many downstream effectors, eventually arresting cellcycle progression and initiating DNA repair. Elements that arrestthe cell cycle include the suppressor of gamma response 1(SOG1) transcription factor, and the cell cycle inhibitory WEE1 ki-nase and SIAMESE-RELATED cyclin-dependent kinase inhibitors

(SMR5 and SMR7) (De Schutter et al., 2007; Yoshiyama et al.,2009; Cools et al., 2011; Yi et al., 2014). Similar to ATR- and ATM-deficient plants, Arabidopsis thaliana plants with knockouts forthese elements display normal vegetative development understandard growth conditions but exhibit growth defects in responseto different kinds of DNA damage. In particular, knockout mutantsof ATR or WEE1 are hypersensitive to replication-inhibitory drugs,demonstrating their importance for the repair of replication stress-induced 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 andSMR7 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 andexchange during meiosis (Humphryes and Hochwagen, 2014;Zamariola, et al., 2014). However, inappropriate HR can produceerroneous DNA rearrangements and intermediate recombinationstructures that cannot be resolved, resulting in genome in-stability (Krejci et al., 2012). Hence, HR must be tightly regulatedand temporally coordinated with cell cycle progression andreplication. In yeast and mammalian cells, several DNA helicasescontribute to HR regulation by unwinding recombination inter-mediates or disrupting Rad51 nucleoprotein filaments (Colavitoet al., 2010). The DNA helicase Regulator of Telomere Length 1(RTEL1) is one of the proteins that suppresses HR through dis-assembling D-loop (two strands of a double-stranded DNA mole-cule separated for a stretch and held apart by a third strand of DNA)recombination intermediates (Barber et al., 2008). Caenorhabditiselegans rtel1 mutants exhibit elevated recombination rates andare synthetically lethal with deletion of the Bloom’s Syndromehelicase (BLM) homolog. This synthetic lethality correlates with

1 Address correspondence to lieven.deveylder@psb.vib-ugent.be.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Lieven De Veylder (lieven.deveylder@psb.vib-ugent.be).www.plantcell.org/cgi/doi/10.1105/tpc.114.134312

The Plant Cell, Vol. 27: 149–161, January 2015, www.plantcell.org ã 2015 American Society of Plant Biologists. All rights reserved.

the accumulation of recombination intermediates that persistand fail to be appropriately repaired. Deficiency of RTEL1 alsoresults in an increased sensitivity to a range of DNA-damagingagents, in particular to DNA interstrand cross-links, which gen-erate lesions affecting replication fork progression (Barber et al.,2008; Uringa et al., 2012).

Apart from suppressing HR, mouse RTEL1 mediates DNAreplication by its association with the proliferating cell nuclearantigen (PCNA) DNA clamp to avoid replication fork stalling orcollapse (Vannier et al., 2013). Conversely, loss of the RTEL1-PCNA interaction is accompanied by replication defects, suchas reduced replication-fork extension rates, increased origin usage,and replication fork instability. In the aspect of telomeres, RTEL1removes telomeric DNA secondary structures, T-loops, and te-lomeric G4-DNA to maintain telomere integrity, and in the ab-sence of RTEL1, a rapid telomere shortening is observed(Vannier et al., 2012). Consistent with the pleiotropic functions ofRTEL1, RTEL1 dysfunction in humans is associated with a rangeof cancers and with Hoyeraal-Hreidarsson syndrome, a rareX-linked recessive disorder (Deng et al., 2013; Vannier et al., 2014).

Despite its importance in mammals, the functions of plantRTEL1 homologs remain to be examined. Here, we report the

characterization of Arabidopsis RTEL1. Analysis of T-DNA in-sertion mutants demonstrated that the plant RTEL1 playsa crucial role in DNA replication, repair, and recombination.Surprisingly, rtel1 mutants display an increased resistance tothe replication inhibitory drug hydroxyurea (HU) that causesa depletion of deoxynucleotide triphosphates (dNTPs). Thisphenotype can be mimicked in wild-type plants by the ad-ministration of DNA interstrand cross-link-inducing agents andis independent of the replication checkpoint regulator WEE1,indicating that RTEL1 deficiency triggers a distinct replicationcheckpoint.

RESULTS

RTEL1KO Plants Exhibit Growth Inhibition Due to CellProliferation Defects

Previous work led to the identification at least two types ofDNA stress, replication defects and DSBs; these activate cellcycle checkpoints, resulting in a cell cycle delay and thus con-tributing to genome stability (Yoshiyama et al., 2013). To furtherdissect 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), andepidermal 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.

150 The Plant Cell

a systematic phenotypic analysis of genes that had been an-notated as putative DNA replication/DNA repair regulators. OneT-DNA insertion line (Salk_113285, hereafter referred to as rtel1-1)exhibited significant growth retardation of the young leaves anddisplayed a smaller final mature leaf size in comparison to wild-type plants (Figures 1A and 1B). Cellular analysis of mature leavesdemonstrated that the decreased leaf area resulted from a re-duction in cell number (Figure 1C), accompanied by a compensa-tory cell enlargement and increase in DNA endoreplication (Figures1D and 1E; Supplemental Figure 1). Similarly, an inhibitory effect onroot growth was observed, showing that rtel1-1 plants havea shorter primary root (Figure 1F). These phenotypes were con-firmed by examination of an independent T-DNA insertion line(Salk_049464, rtel1-2) (Figure 1). In both lines, a reduction in rootmeristem size was observed (Figure 1G), correlated with a decreasein the number of cortical meristem cells (45.3 6 2.5 in wild-typeplants 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 compensa-tory increase in mature cell length (198.4 6 3.8 mm in wild-typeplants versus 253.8 6 4.3 mm and 223.5 6 13.2 mm in rtel1-1 andrtel1-2, respectively [n > 100]). Thus, the observed root growthdefect of the RTEL1 deficient plants is primarily due to an inhibitionof cell division, rather than cell elongation.

The T-DNA insertions in rtel1-1 and rtel1-2 are in the 7th exonand 16th intron of the RTEL1 gene (At1g77950), respectively(Supplemental Figure 2A). RT-PCR failed to detect full-lengthRTEL1 transcripts in the two mutants, suggesting that they arenull (Supplemental Figure 2B). Through a BLAST search usingthe amino acid sequence of Arabidopsis RTEL1, we found 22

Figure 3. Depletion of RTEL1 Leads to Replication Defects.

(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 typewere arbitrarily set to one. All values were normalized against the ex-pression 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, **Pvalue < 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 sizebased on the cortical cell length. Bars = 50 mm.

Figure 2. Deficiency of Arabidopsis RTEL1 Does Not Cause Telomere Loss.

(A) Telomere length comparison between wild type (Col-0) and rtel1mutantsof the 2nd generation. Genomic DNA was isolated from 8-d-old seedlingsand digested with the restriction enzyme TruII. DNA gel blot analysis wasperformed using a digoxigenin-labeled telomere repeat as probe.(B) Morphology of 3-week-old wild type (Col-0), rtel1-1, stn1-1, andrtel1-1 stn1-1. Plants were isolated from an F2 segregating populationgenerated by crossing rtel1-1 with stn1-1. Bars = 1 cm.

RTEL1 Suppresses Replication Stress 151

protein sequences with a high similarity to At-RTEL1 across sixmodel species (Arabidopsis, Oryza sativa, Saccharomycescerevisiae, C. elegans, Mus musculus, and Homo sapiens). Phylo-genetic analysis showed that they classify into four groups, withAt1g79950 falling into the RTEL1 group (SupplementalFigure 3A). Conserved domain analysis showed that Arabi-dopsis RTEL1 contains a Rad3-related DNA helicase (RAD3)domain, a PIP-box like domain, and a harmonin-N-like do-main (Supplemental Figure 3B). Compared with Hs-RTEL1and Mm-RTEL1, At-RTEL1 lacks one harmonin-N-like do-main and a C4C4 domain or a RING-finger domain, indicatingthat the Arabidopsis orthologous gene might have lost a partof its gene functions (Supplemental Figure 3B).

RTEL1 Deficiency Does Not Result in Telomere Shortening

RTEL1 was originally characterized as a regulator of telomerelength in mice, with embryonic stem cells deficient in RTEL1 dis-playing short telomeres (Ding et al., 2004). To test whether plantRTEL1 deficiency also causes telomere loss, we performed a ter-minal restriction fragments analysis on wild type, rtel1-1, and rtel1-2mutants. Unexpectedly, all genotypes displayed identical telomerelengths (Figure 2A). Moreover, in contrast to known telomere mu-tants (Fitzgerald et al., 1999; Mozgová et al., 2010), no progressiveshortening 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 withthe CST complex, we crossed rtel1-1 with stn1-1 and analyzed theF2 segregating population. As expected, both rtel1-1 and stn1-1single mutants showed smaller leaves compared with wild-typeplants (Figure 2B). The rtel1-1 stn1-1 double mutant exhibited de-formed leaves (Figure 2B). Unexpectedly, the telomere length ofstn1-1 rtel1-1 double mutants was not reduced but rather elon-gated compared with stn1-1 single mutants (Supplemental Figure4B). Thus, the observed growth phenotypes observed for the rtel1mutants are unlikely to originate because of telomere defects.

Depletion of RTEL1 Causes Replication Defects

To understand the mechanism underlying the observedgrowth reduction of RTEL1KO plants, an RNA sequencingexperiment was conducted, comparing the transcriptome ofrtel1-1 and Columbia-0 (Col-0) root meristems. In total, 505

genes were differentially regulated (fold change $ 1.5; Pvalue # 0.01), with 407 genes upregulated and 98 genesdownregulated (Supplemental Data Set 1). Among a set of 61genes identified before as DNA stress hallmark genes (Yiet al., 2014), 46 transcripts (75%) were upregulated in rtel1-1mutant root meristems (Supplemental Table 1). In accor-dance, Gene Ontology (GO) analysis showed among theupregulated genes a significant overrepresentation of tran-scripts associated with response to ionizing radiation andDSB repair (Supplemental Figure 5). Transcriptional acti-vation of DNA damage marker genes (such as PARP2,CYLINB1;1, and BRCA1) in rtel1 mutant seedlings wasconfirmed through a quantitative PCR approach (Figure 3A).These results indicated that RTEL1 deficiency generatesdamaged DNA.Based on the interaction of the mouse RTEL1 protein with PCNA

(Vannier et al., 2013), we reasoned that the observed expression ofDNA damage marker genes in rtel1-1 might be caused by prob-lematic DNA replication. To confirm this hypothesis, we estimatedthe duration of the S-phase through measurement of the in-corporation rate of ethynyl deoxyuridine (EdU), a thymidine analog

Table 1. MMC Administration Exhibits a Positive Correlation with RTEL1 Deficiency at the Transcriptional Level

Col-0 sog1-1 MMC-Col-0 MMC-sog1-1 rtel1-1 rtel1-1 sog1-1

BRCA1 1.00 6 0.006 0.95 6 0.118 3.91 6 0.244 1.83 6 0.124 5.15 6 0.047 1.08 6 0.011PARP2 1.00 6 0.057 0.81 6 0.013 4.87 6 0.199 2.44 6 0.171 4.55 6 0.045 1.40 6 0.038RAD51 1.00 6 0.030 0.94 6 0.027 2.63 6 0.380 1.31 6 0.032 2.88 6 0.097 1.02 6 0.078RNR1 1.00 6 0.034 0.92 6 0.003 1.83 6 0.151 1.38 6 0.102 1.81 6 0.064 0.98 6 0.083SMR7 1.00 6 0.029 0.95 6 0.068 15.28 6 2.418 4.58 6 1.210 15.76 6 0.235 1.69 6 0.178TSO2 1.00 6 0.063 0.97 6 0.013 4.10 6 0.338 1.39 6 0.046 3.23 6 0.227 0.62 6 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 supplementedwith 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 thereferences genes. Data represent mean 6 SE (n = 2).

Figure 4. RTEL1 Deficiency Triggers Increased HR.

Recombination frequencies of wild-type (Control) and rtel1-1 seedlingsusing the 651 or IC9C reporters. Data represent mean number of GUSsectors 6 SD (n = 4, minimum 50 plants per repeat).

152 The Plant Cell

that can be incorporated into genomic DNA during replication(Hayashi et al., 2013). S-phase duration in rtel1-1 (4.21 h) was sig-nificantly prolonged in comparison with that in the wild type (2.01 h).To further emphasize that RTEL1-deficient plants suffer fromproblematic DNA replication, we introduced the rtel1-1 mutationinto atr-2 and wee1-1 plants, which are hypersensitive to repli-cation stress. Primary roots of rtel1-1 atr-2 and rtel1-1 wee1-1double mutants were significantly shorter than those of rtel1-1plants (Figures 3B and 3C) accompanied with shrinkage of theirmeristems (Figures 3D and 3E), indicating that both WEE1 andATR suppress the rtel1 mutant phenotype.

RTEL1 Suppresses HR

As the HRmarker geneRAD51was highly induced in rtel1-1mutants(Table 1; Supplemental Data Set 1), we postulated that RTEL1deficiency might result in increased HR. To test this, we in-troduced two different HR substrates, 651 and IC9C, intothe rtel1-1 background. Both substrate lines harbor two

nonfunctional parts of the b-glucuronidase (GUS) gene (uidA)in a spatial orientation that can be restored into a functionaluidA gene either by intra- and interchromosomal re-combination in the 651 line or by only interchromosomalrecombination in the IC9C line (Swoboda et al., 1994; Molinieret al., 2004; Schuermann et al., 2005). Both reporters re-vealed a significant increase in the level of spontaneous HRevents in rtel1-1, compared with wild-type plants (Figure 4).The Arabidopsis MUS81 endonuclease is crucial for the res-olution of Holliday junctions induced by HR (Hartung et al.,2006; Berchowitz et al., 2007). A synergistic effect on plantgrowth of rtel1-1 with mus81-1 was observed, since doublemutants displayed a shorter root length and root meristem,compared with the mus81-1 single mutants that are pheno-typically indistinguishable from wild-type plants (Figures 5Ato 5C), confirming the increase in HR events in the rtel1background.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.

RTEL1 Suppresses Replication Stress 153

were observed in RECQ4A-deficient plants (Hartung et al.,2007). To define the genetic interaction between RECQ4A andRTEL1, we generated the rtel1-1 recq4a-4 double mutant.The recq4a-4 single mutant develops normally under non-stress conditions, albeit showing hypersensitivity to DNA

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

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-producingagents 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 wasmeasured 7 d after sowing. Data represent mean 6 SE (n > 10).

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 sizebased on the cortical cell length. Bar = 50 mm.

154 The Plant Cell

RTEL1KO Mutants Are Hypersensitive to DNACross-Linking Agents

Increased HR in rtel1 mutants suggested that RTEL1 deficiencymay trigger DNA damage. To verify this, we determined the sen-sitivity of rtel1mutants to genotoxic agents. Depletion of RTEL1 didnot alter the sensitivity to bleomycin and zeocin, which triggerDSBs (Figure 6A). In contrast, rtel1 mutants were hypersensitive tomitomycin C (MMC) and cis-platin (CP), which trigger DNA cross-linking (Figure 6B). MMC mainly generates interstrand cross-linkson DNA, whereas CP preferentially forms intrastrand cross-links.These results point out that RTEL1 has a function in the repair ofcross-linked DNA.

Based on the observed effect of RTEL1 deficiency on S-phaseprogression and suppression of the root growth phenotype by bothATR and WEE1 (Figures 3B to 3E), it is likely that cross-linked DNAmay interfere with the progression of the replication fork. Replica-tion 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 weretolerant to HU, as observed by a decreased reduction inroot growth, compared with control plants (Figure 6C).Cellular analysis illustrated that the HU tolerance pheno-type of the rtel1-1 mutant plants resulted from maintenance

of meristem cell number rather than an increase in celllength (Supplemental Figure 6).The HU tolerance induced by RTEL1 deficiency was even more

pronounced in the WEE1- or ATR-deficient background (Figure7A). Microscopy analysis confirmed that the observed growthdifferences were attributed to changes in meristem length (Figures7B to 7D). Strikingly, whereas the meristems of the wee1-1 andatr-2 mutants were strongly affected by HU treatment due to thelack of the activation of a replication checkpoint, this meristemphenotype was strongly suppressed by the rtel1mutation (Figures7B to 7D), confirming that RTEL1 deficiency triggers a cell cyclecheckpoint abrogating the need for ATR and WEE1.

DNA Cross-Linking Agents Phenocopy the HU ResistancePhenotype Caused by RTEL1 Depletion

Our aforementioned results suggested that the occurrence ofcross-linked DNA might confer HU resistance. To test this hy-pothesis, we applied the DNA cross-linking drugs CP and MMC towild-type plants. Similar to rtel1 mutants, both MMC-treated andCP-treated plants became tolerant to HU, as observed by a lesspronounced reduction in root growth compared with that of con-trol plants (Figures 8A and 8D) along with the absence of shrinkageof 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) treatedwithout (-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 sizebased on the cortical cell length. Bar = 50 mm.

RTEL1 Suppresses Replication Stress 155

was more pronounced for the HU-hypersensitive wee1-1 plants(Figures 8A and 8D), in which MMC or CP treatment rescued thestrong effect of HU on meristem size (Figures 8C and 8F). Theseresults support the idea that the type of damage induced by RTEL1deficiency mimics that caused by cross-linked DNA, a hypothesissubstantiated by quantitative PCR, demonstrating that genes beingtranscriptionally induced in rtel1mutants show a similar activation asin wild-type plants treated with MMC (Table 1).

The HU Resistance Phenotype Triggered by Cross-LinkedDNA Depends on SOG1

Because the replication checkpoint induced by RTEL1 de-ficiency or DNA cross-linking agents appeared to be WEE1- and

Figure 9. The HU Resistance Phenotype Induced by Cross-Linked DNARequires Functional SOG1, but Not ATM.

(A) Root growth of 7-d-old atm-1, wee1-1, wee1-1 atm-1, sog1-1, andwee1-1 sog1-1 mutants grown on control medium (-HU), medium sup-plemented with 2.5 mg/L MMC, 0.5 mM HU, or 2.5 mg/L MMC + 0.5 mMHU (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 me-dium (-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-sidedStudent’s t test).

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

(A) to (C) Root growth of 7-d-old wild type (Col-0), wee1-1, sog1-1, andwee1-1 sog1-1 mutants grown on control medium (-HU) or mediumsupplemented with 0.5 mM HU (+HU). Bar = 5 mm.(B) Quantification of root length of plants shown in (A). Data representmean 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 sizebased on the cortical cell length. Bar = 50 mm.

156 The Plant Cell

ATR-independent (Figures 7 and 8), we tested whether the res-cuing signaling pathway might depend on ATM and SOG1. Totest this hypothesis, we generated atm-1 wee1-1 and sog1-1wee1-1 double mutants because the MMC-conferring phenotypeis clearer in the background of wee1-1 than that of wild-typeplants. Plants were treated with MMC only or in combination withHU. Similar to wee1-1 single mutants, MMC administration toatm-1 single and atm-1 wee1-1 double mutants resulted in HUresistance, showing that the rescuing pathway does not rely onfunctional ATM (Figure 9A). By contrast, sog1-1 and sog1-1wee1-1 mutants lost the HU resistance phenotype (Figure 9A).Similar to MMC administration, HU resistance induced by RTEL1deficiency was also deprived in both sog1-1 and sog1-1 wee1-1(Figure 9B). Additionally, quantitative PCR experiments confirmedthat the expression of DNA stress genes induced by RTEL1 de-ficiency or MMC treatment depended predominantly on the SOG1transcription factor (Table 1).

The data obtained indicated that next to WEE1, SOG1 might bean essential replication checkpoint regulator. To confirm this hy-pothesis, 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 visualizationshowed that sog1-1 root meristems exhibited a cell death pheno-type under HU treatment, similar as that reported before for wee1-1mutant plants (Figure 10C). Surprisingly, compared with the sog1-1and wee1-1 single mutants, sog1-1 wee1-1 plants were extremelyHU sensitive, with the sog1-1 wee1-1 double mutant root lengthbeing only 23.6 and 10.4% of wee1-1 and sog1-1 roots, re-spectively (Figure 10B). Moreover, in the sog1-1 wee1-1 doublemutants, the meristem was totally consumed under replicationstress conditions (Figure 10C). These data illustrate that SOG1 andWEE1 independently control the replication stress checkpoint.

DISCUSSION

In mammals and nematodes, RTEL1 controls different functionsrelated to genome integrity (Ding et al., 2004; Vannier et al., 2014).Here, we show that deficiency of the Arabidopsis RTEL1 proteinaffects DNA replication, repair, and recombination, but we ob-served no obvious role in the control of telomere length. Thesedata indicate that RTEL1’s role in DNA replication might beconserved but that the plant ortholog might have lost its role intelomere regulation. Correspondingly, plant RTEL1 proteins lackthe C4C4 domain, whose mutation in the human RTEL1 results inelevated telomere loss (Le Guen et al., 2013; Vannier et al., 2014).Although phylogenetic analysis showed that Arabidopsis carriesonly one copy of the RTEL1 gene, supported by the phenotypicdefects observed for the knockout plants, it cannot be excludedthat RTEL1 might exhibit functional redundancy with its homologsin the regulation of telomere integrity. One particular candidategene is FANCJ (At1g20750), whose expression is strongly inducedin rtel1-1 mutants.

Surprisingly, introducing the rtel1-1 mutation into an stn1-1mutant background resulted in an increased telomere length,rather than the expected shortening. STN1 associates withTEN1 and CDC13 to form a trimeric complex that is critical for

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 triggersthe ATM/ATR-mediated DNA damage response. We postulatethat the effects of the mutation of rtel1-1 on DNA replication andHR might enhance the stringency of the activated cell cyclecheckpoint pathways, with a synergistic growth inhibitory effect asa consequence. Simultaneously, as ATR contributes to telomerelength maintenance (Amiard et al., 2011; Boltz et al., 2012), thertel1-1 activated ATR pathway might account for the observedpartial 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 attributedto DNA replicative defects, triggering a cell cycle checkpoint toarrest DNA replication and cell cycle progression. In support ofthis, rtel1 mutants exhibited a prolonged S-phase, expression ofDNA damage response genes, and synergetic growth defects withATR- and WEE1-deficient plants, suggesting that ArabidopsisRTEL1 participates in DNA replication. In agreement, mouse RTEL1associates with the replisome through binding to PCNA through itsPIP box, which is conserved in the plant protein. Accordingly,disruption of the RTEL1-PCNA interaction compromises replicationfork stability and slows down replication, inducing growth arrestand cell senescence (Vannier et al., 2013).

Figure 11. Model for Replication Checkpoint Activation in Plants.

HU treatment results in stalled replication forks that activate the WEE1kinase in an ATR-dependent manner. By contrast, DNA cross-links in-duced byMMC treatment or absence of RTEL1 induce a SOG1-dependentinhibition of cell cycle progression, probably involving MAP kinases andthe CDK inhibitor SMR proteins. Indirectly, the DNA cross-links may pre-activate a replication checkpoint that confers HU resistance.

RTEL1 Suppresses Replication Stress 157

In addition to replication defects, the absence of RTEL1caused increased HR, supported by the phenotype of rtel1-1mus81-1 double mutants. MUS81 is a highly conserved endonu-clease and associates with the EME1 endonuclease to resolve HRintermediates, such as 39 flap structures and Holliday-like DNAjunctions. The synergetic defects of rtel1-1 and mus81-1 indicatethat the resolution of the HR intermediates arising from RTEL1deficiency requires the MUS81/EME1 complex (Hartung et al.,2006; Mannuss et al., 2010). This is consistent with observations inC. 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 inrtel1-1 could at least partially result from replicative defects. Similarto the human RTEL1 helicase, the Arabidopsis RTEL1 may directlysuppress HR by unwinding heteroduplex DNA (D-loop) (Barberet al., 2008). The synthetic lethality of rtel1-1 with recq4a-4 illus-trates that the HR repressing function of RTEL1 differs from that ofSGS1/BLM, which prevents the formation of multichromatid jointmolecules and dissolves double Holliday junctions in S. cerevisiae(Oh et al., 2007; Bernstein et al., 2010). Thus, plants appear to usedifferent helicases to resolve distinct types of DNA structures thatarise during replication, repair, and recombination.

The increased sensitivity of rtel1 mutants to MMC and CPfurther supports RTEL1’s role in DNA replication and HR. CPand MMC induce intrastrand cross-links and interstrand cross-links, respectively. The lack of sensitivity to bleomycin andzeocin, by which DSBs are generated, indicates that RTEL1 isspecifically required for the repair events of DNA cross-links.Intriguingly, rtel1 mutant seedlings are more tolerant to HUcompared with wild-type plants. Moreover, MMC and CP ap-plication mimic the HU resistance phenotype of rtel1-1 mutants,indicating that cross-linked DNA accounts for the observed HUresistance. HU is a direct inhibitor of ribonucleotide reductase(RNR) and its presence depletes the dNTP pool of cells, causinga slowdown of replication fork progression and a reduced acti-vation of replication origins (Wang and Liu, 2006; Saban and Bujak,2009). One mechanism to explain the observed HU resistancephenotype might be an upregulation of RNR expression, alleviatingthe dNTP drop. Alternatively, rtel1-induced DNA damage mightpreactivate a DNA damage response that confers HU resistance.DNA replication consists out of two steps: DNA unwinding byhelicases and subsequent synthesis of the new DNA strands byDNA polymerases. The DNA replication checkpoint coordinatesthese two processes under the control of ATR. If dNTP levels arereduced, plants with a functional checkpoint will try to match theirDNA helicase activity with the maximal polymerase activity giventhe available dNTP pool, e.g., by preventing the activation of neworigins of replication. We have speculated before that the HU-hy-persensitive phenotype of WEE1-deficient plants arises because oftheir inability to coordinate the DNA replication rate with dNTPavailability. This likely results in long stretches of single-strandedDNA that may become artificial substrates for homologous re-combination, resulting in DNA deletions (Kalhorzadeh et al., 2014).It can be easily envisioned that the arrest of DNA helicases by DNAcross-links, arising through RTEL1 deficiency or MMC treatment,may counteract the unwinding of DNA and thus elevate the needfor WEE1 (Figure 11). As a result, long stretches of single-strandedDNA will not occur and only the repair of the cross-links is required.

Our genetic analysis indicates that this repair pathway depends ona functional SOG1, as the HU resistance phenotype was lost inrtel1 sog1-1 double mutant plants. Thus, next to its demonstratedrole in sensing DSBs (Yoshiyama et al., 2009), SOG1 appears tocontrol a replication-dependent checkpoint in response to DNAcross-links. The WEE1-dependent and SOG1-dependent path-ways appear to be engaged upon the occurrence of stalled repli-cation forks, based on the HU hypersensitive phenotype of thesog1-1 wee1-1 double mutants, in comparison to the sog1-1 andwee1-1 single mutants. Under these conditions, SOG1 is likely tobe 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 twofamily members of the recently described class of SIAMESE-RELATED cyclin-dependent kinase inhibitors, of which at leastSMR7 is under direct transcriptional control of SOG1 (Yi et al.,2014), since both genes show a strong transcriptional induction inRTEL1-deficient plants.Since DNA stress does not induce SOG1 transcription, SOG1

protein activity might be controlled at the posttranscriptionallevel. Three proteins from the mitogen-activated protein (MAP)kinase pathway, MAP kinase 3 (MPK3), MPK6, and MAP kinasephosphatase 1, were previously linked with DNA stress, namely,methyl methanesulfonate treatment and UV radiation, and operateindependently from ATR (González Besteiro et al., 2011; GonzálezBesteiro and Ulm, 2013). Since the DNA cross-linking pathway isnot controlled by ATR or ATM, it is an intriguing possibility that theMAP 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 DNAcross-linking agents, illustrating an important role for the RTEL1helicase in resolving aberrant replication structures. More-over, next to its previously recognized role in DSB checkpointcontrol, our data identified the SOG1 transcription factor as anessential replication stress checkpoint regulator, working in-dependently from WEE1. Our data illustrate that distinct types ofDNA damages employ different signaling pathways to arrestDNA synthesis upon the occurrence of replication defects.

METHODS

Plant Materials and Growth Conditions

Arabidopsis thaliana plants were grown under long-day conditions (16 h oflight/8 h of darkness) at 22°C on half-strength Murashige and Skoog (MS)germinationmedium (MurashigeandSkoog, 1962). The rtel1-1 (SALK_113285)and rtel1-2 (SALK_046494) alleleswereacquired from theABRC.Homozygousinsertion alleles were checked by genotyping PCR using the primers listed inSupplemental Table 4. The atm-1, atr-2, wee1-1, sog1-1, mus81-1, and re-cq4a-4 mutants have been described previously (Garcia et al., 2003; Culliganet al., 2004; De Schutter et al., 2007; Yoshiyama et al., 2009; Hartung et al.,2006, 2007;Chenet al., 2008). For the treatmentswithgenotoxic agents, seedswere directly plated on control medium and medium supplemented with theindicated 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 clearedovernight in ethanol and stored in lactic acid. The leaf area, the cell area of

158 The Plant Cell

pavement cells, and the total number of cells per leaf were obtained asdescribed previously (Yi et al., 2014). For root meristem observations, roottips of 7-d-old seedlings were stained for 3 min in a 10 mM propidium iodidesolution (Sigma-Aldrich), washed for 15minwithMilli-Qwater, and visualizedwith an LSM 5 exciter confocal microscope (Zeiss). For flow cytometricanalysis, leaf material was chopped in 200 mL of Cystain UV Precise P nucleiextraction buffer (Partec), supplemented with 800 mL of staining buffer. Thefiltered supernatants were measured by the Cyflow MB flow cytometer(Partec). The nuclei were analyzed with the CyFlow flow cytometer usingFloMax (Partec) software.

Phylogenetic Tree Construction and Conserved Domains Analysis

RTEL1 homologs were identified from GenBank using the protein basiclocal alignment search tool (BLASTp) (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The alignment of full-length amino acid sequences was used toconstruct the neighbor-joining tree using the MEGA3 (Molecular Evolu-tionary Genetic Analyses, version1.1, Pennsylvania State University; http://www.megasoftware.net/) package. Conserved domains of ArabidopsisRTEL1 were predicted based on the protein structure of human and mouseRTEL1 homologs described by Vannier et al. (2014).

RNA Sequencing and GO Analysis

Root tips (<2 to 3mm) of 7-d-old seedling of thewild type (Col-0) and rtel1-1were collected and frozen in liquid nitrogen. The resulting samples wereused for RNA extraction with the plant RNeasy kit (Qiagen). Illumina True-Seq libraries, quality of the reads (Phred quality score), and empiricalanalysis of gene expression data were performed as described (Kalhorzadehet al., 2014). To determine significantly overrepresented GO categoriesamong upregulated genes, the BiNGO plug-in for Cytoscape was used(http://www.psb.ugent.be/cbd/papers/BiNGO/) (Maere et al., 2005).

Telomere Length Analysis

Genomic DNA was isolated using the DNeasy plant kit (Qiagen) anddigested with restriction enzyme TruII for measuring telomere length. Thehybridization probe was prepared using the DIG oligonucleotide 39-endlabeling kit (2nd generation). Hybridization and detection were performedaccording to the manufacturer’s instructions (Roche Applied Science).

Quantitative RT-PCR

RNA was extracted from root tips of 8-d-old seedlings with the RNeasyplant kit (Qiagen), and cDNA was prepared from 1 µg of total RNA with theiScript cDNA synthesis kit (Bio-Rad) according to the manufacturer’sinstructions. Quantitative RT-PCR was performed with LightCycler 480SYBR Green I Master (Roche) in a final volume of 5 mL and analyzed witha LightCycler 480 (Roche). For each reaction, three technical repeatsand two to three biological repeats were done. The expression level ofeach gene was normalized by the following reference genes: ACTIN2,EMB2386, PAC1, and RPS26C. Primers used for the RT-PCR are given inSupplemental Table 2.

HR Assay

Two recombination substrates, 651 (Swoboda et al., 1994) and IC9C(Molinier et al., 2004), were crossed into rtel1-1. For the HR recombinationassay, 50 seeds of each line were germinated on half-strength MS. Therestoration of the reporter gene was visualized by histochemical GUSstaining according to the standard protocol (Jefferson et al., 1987). HRevents of individual plants were assessed visually using a binocularmicroscope. The HR assays were repeated three times, and the meanvalues were calculated.

S-Phase Duration Evaluation

Four-day-old seedlings grown vertically on half-strength MS were transferredto liquid medium (0.53 MS, 1% sucrose, and 10 mM EdU in Click-iT com-ponent 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-phaseevaluation 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 datafrom this article can be found in the Arabidopsis Genome Initiative orGenBank/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.

Supplemental Figure 3. Phylogenetic Analysis of RTEL1.

Supplemental Figure 4. Arabidopsis RTEL1KO Does Not CauseTelomere Loss.

Supplemental Figure 5. GO Analysis of the Upregulated Genes in theRoot Tips of rtel1 Mutant Plants.

Supplemental Figure 6. Cellular Analysis of Wild-Type and rtel1-1 Roots.

Supplemental Table 1. DNA Stress Hallmark Genes Induced in rtel1-1Compared with the Wild Type.

Supplemental Table 2. Primers Used for Genotyping and RT-PCR.

Supplemental Data Set 1. Up- and Downregulated Genes in rtel1-1Compared with the Wild Type (Col-0).

Supplemental Data Set 2. Text File of Alignment Corresponding tothe Phylogenetic Analysis in Supplemental Figure 3.

ACKNOWLEDGMENTS

We thank Annick Bleys for help preparing the article. This work wassupported by grants of the Research Foundation Flanders (G.0C72.14N)and the Interuniversity Attraction Poles Programme (IUAP P7/29 “MARS”),initiated by the Belgian Science Policy Office. T.C. is a Postdoctoral Fellowof the Research Foundation-Flanders.

AUTHOR CONTRIBUTIONS

Z.H. and L.D.V. conceived and designed the research. Z.H., T.C., J.H., andP.K. performed the experiments. Z.H., T.C., J.H., and L.D.V. analyzed the dataand wrote the article. All authors read, revised, and approved the article.

Received November 14, 2014; revised December 17, 2014; acceptedJanuary 6, 2015; published January 16, 2015.

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RTEL1 Suppresses Replication Stress 161

DOI 10.1105/tpc.114.134312; originally published online January 16, 2015; 2015;27;149-161Plant Cell

Zhubing Hu, Toon Cools, Pooneh Kalhorzadeh, Jefri Heyman and Lieven De VeylderCheckpoint in Response to DNA Cross-Links

Helicase RTEL1 Triggers a SOG1-Dependent ReplicationArabidopsisDeficiency of the

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