Proteins of the Fanconi Anemia (FA) complementation group are required for crosslink (CL) repair in humans and their loss leads to severe pathological phenotypes. Here we characterize a homolog of the Fe-S cluster helicase FANCJ in the model plant Arabidopsis, AtFANCJB, and show that it is involved in interstrand CL repair. It acts at a presumably early step in concert with the nuclease FAN1 but independently of the nuclease AtMUS81, and is epistatic to both error-prone and error-free post-replicative repair in Arabidopsis. The simultaneous knock out of FANCJB and the Fe-S cluster helicase RTEL1 leads to induced cell death in root meristems, indicating an important role of the enzymes in replicative DNA repair. Surprisingly, we found that AtFANCJB is involved in safeguarding rDNA stability in plants. In the absence of AtRTEL1 and AtFANCJB, we detected a synergetic reduction to about one third of the original number of 45S rDNA copies. It is tempting to speculate that the detected rDNA instability might be due to deficiencies in G-quadruplex structure resolution and might thus contribute to pathological phenotypes of certain human genetic diseases.
The Fanconi Anemia and Hoyeraal-Hreidarsson syndromes are severe human genetic diseases that are correlated with DNA repair deficiencies. Interestingly, plants harbour homologues of factors in their genome that are causative for the respective diseases upon mutation. Using the model plant Arabidopsis, we analysed in detail the role of two helicases that are causing one or the other syndrome when defective in humans. We found that the simultaneous loss of both helicases leads to synergistic defects of plant cells in response to induced but also natural DNA damage, indicating that they act in different repair pathways. Most surprisingly, we found a drastic reduction of rDNA copies, in the double mutant, that are required for ribosome formation and thus protein synthesis. Such a phenotype has not been reported before—neither for humans nor animals—but this finding might significantly contribute to specificities of the respective diseases.
Citation: Dorn A, Feller L, Castri D, Röhrig S, Enderle J, Herrmann NJ, et al. (2019) An Arabidopsis FANCJ helicase homologue is required for DNA crosslink repair and rDNA repeat stability. PLoS Genet 15(5): e1008174. https://doi.org/10.1371/journal.pgen.1008174
Editor: Avraham Levy, Weizmann Institute, ISRAEL
Received: March 26, 2019; Accepted: May 3, 2019; Published: May 23, 2019
Copyright: © 2019 Dorn et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: Sequence data from this article can be found with the following Arabidopsis AGI locus identifiers, AtFANCJA: At1g20750, AtFANCJB: At1g20720, AtFAN1: At1g48360, AtRECQ4A: At1g48360, AtMUS81: At4g30870, AtRAD5A: At5g22750, AtREV3: At1g67500, AtRTEL1: At1g79950.
Funding: This work was supported by the European Research Council ERC (Grant numbers ERC-2010-AdG_20100317 COMREC and ERC-2016-AdG_741306 CRISBREED to HP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The helicase FANCJ was initially identified in humans as a direct interaction partner of the tumour suppressor protein BRCA1, thereby originally being named BACH1 (BRCA1-associated C-terminal helicase 1) or BRIP1 (BRCA1 interacting protein 1) . The helicase was later determined as the causal gene of the Fanconi Anemia complementation group J [2–4]. Fanconi Anemia (FA) is a rare human hereditary disease, characterized by bone marrow failure and a high cancer predisposition . At least 22 genes were identified in causing FA upon mutation, including the two helicases FANCM and FANCJ . On a cellular level, FA patients exhibit a characteristic hypersensitivity against DNA crosslinking agents such as Mitomycin C (MMC), hinting to the important function of the FA proteins in interstrand-crosslink (ICL) repair . ICLs pose a serious threat to genome stability as they connect both strands of the DNA double helix, thereby blocking replication and transcription. The FANC genes cooperate in the human canonical ICL repair pathway. Its exact repair mechanism is still under investigation, but damage recognition relies on the helicase FANCM. FANCM, together with at least eight further FA proteins, forms the FA core complex that initiates the monoubiquitination of the FANCD2/I heterodimer. This leads to the recruitment of a multitude of ICL repair factors, catalysing the nucleolytic excision of the crosslink (unhooking), resulting in a one-sided double strand break (DSB). The DSB is repaired via homologous recombination, while translesion synthesis (TLS) bypasses the unhooked crosslink, which can be removed by further processing by nucleotide excision repair (NER) factors . The FA factor FANCJ is a Fe-S cluster helicase with 5’-3’ directionality, acting on dsDNA substrates with 5’ ssDNA overhangs, but also unwinds D-Loops without single-stranded regions [8,9]. The exact function of FANCJ in the FA pathway is still obscure, but it was shown that the helicase is not necessary for FANCD2 monoubiquitination, indicating a possible later involvement . FANCJ was demonstrated to act together with its interaction partner BRCA1 in promoting HR following DSBs, however FANCJ also has a destabilizing function on RAD51 nucleoprotein filaments [10,11]. Furthermore, a role for FANCJ besides from the FA pathway, has been postulated with a function in the removal of G-quadruplex (G4) DNA structures[12,13]. Interestingly, a direct interaction of FANCJ and the RecQ helicase BLM was shown, and both helicases seem to cooperate in response to replicative stress [14,15]. These various functions hint to a multifunctional role of FANCJ in the maintenance of genome stability. In the plant model organism Arabidopsis thaliana, only a small subset of FA factors could be identified so far, with the helicase FANCM being the only plant FA protein involved in CL repair . However, AtFANCD2 and AtFANCM have been shown to be involved in meiotic recombination [16–18]. Nevertheless, CL repair functions could be described for Arabidopsis homologs of the FA-associated proteins FAN1 and MHF1 [19,20]. Although two Arabidopsis FANCJ paralogs had been identified by our group several years ago, further characterization had not been carried out till now .
Closely related to FANCJ is a further Fe-S cluster helicase, RTEL1, for which an involvement in G4 DNA metabolism was also reported . Mutations in RTEL1 lead to Hoyeraal-Hreidarsson syndrome in humans, a severe form of dyskeratosis congenital that is characterized by bone marrow failure, immunodeficiency and growth retardation [23–26]. Mammalian RTEL1 was shown to act in the preservation of telomere stability, as it dissolves T-Loops and telomeric G4 DNA structures, furthermore loss-of-function mutations lead to telomere-loss and chromosome fusions [22,27]. Thereby a parallel involvement of RTEL1 and the RecQ helicase BLM could be shown. In C. elegans, a role for RTEL1 in ICL repair was demonstrated, furthermore the helicase was characterized as a antirecombinase that suppresses recombination by disassembling D-Loops . Interestingly, simultaneous loss of RTEL1 and the FANCJ homolog here lead to synthetic lethality, indicating independent essential roles for both helicases . For the Arabidopsis RTEL1 homolog, its antirecombinogenic function, as well as its involvement in CL repair, was demonstrated [21,29]. The RTEL1 mediated suppression of HR defines a pathway independent of the FA helicase FANCM or the RTR complex [21,30]. AtRTEL1 seems to fulfil multifaceted functions in stabilizing the genome, with a role in telomere homeostasis but also a role in the maintenance of 45S rDNA repeat number parallel to the RTR complex being shown [21,30]. This highlights the importance of RTEL1 in preserving repetitive sequences within the plant genome.
As the classical FA pathway for ICL repair does not seem to be conserved in plants, we set out to characterize their specific network of crosslink repair factors over the last recent years. Initially, a three-branched model of CL repair, defined by the RecQ helicase RECQ4A, the nuclease MUS81 and the ATPase RAD5A, has been proposed . AtRECQ4A is the functional homolog of the human BLM helicase and part of the evolutionary conserved RTR complex [32,33]. RECQ4A was shown to act in ICL repair in a common pathway with the FA-associated nuclease FAN1 but independently of FANCM [19,20]. The ATPase AtRAD5A is the functional homolog of yeast Rad5 and acts in the error-free branch of post-replicative repair (PRR). For the Arabidopsis homolog, a function in DNA repair, apart from other pathways such as nucleotide excision repair, single-strand break repair, micro-homology mediated double-strand break repair and the ATM-mediated DNA damage response, was demonstrated [34–36]. The endonuclease MUS81 seems to mediate a separate backup pathway for the resolution of repair intermediates, but interestingly acts in a common pathway with the helicase RTEL1 . Furthermore, a parallel involvement of MUS81 and FAN1 in CL repair was shown. As double mutants of MUS81 with the RTR complex partners RECQ4A and TOP3α or the FA helicase FANCM exhibit synthetic lethality, this further emphasizes the importance of these parallel pathways in the repair of replicative DNA damage [20,37,38]. MUS81 was only recently identified as the central player in DNA-protein crosslink repair, where the nuclease acts in parallel to the protease WSS1A and the tyrosyl-DNA-phosphodiesterase TDP1 . REV3, the catalytic subunit of the translesion synthesis (TLS) DNA polymerase Zeta is an additional important player in ICL repair, as TLS acts downstream of the FA pathway and in the error-prone post-replicative repair . Mutants for the Arabidopsis homolog AtREV3 were also shown to be sensitive towards MMC induced DNA damage, indicating conserved functions .
We now wanted to characterize the role of FANCJ homologs from Arabidopsis in ICL repair for the first time, thereby aiming to include them into the CL repair network. Surprisingly, only one FANCJ homolog, AtFANCJB, turned out to possess a function in ICL repair, but also, an exciting role of this homolog in the maintenance of 45S rDNA repeat stability in parallel to the related helicase RTEL1 could be revealed.
AtFANCJB is involved in ICL repair but not in the suppression of HR
Through BLAST analyses with the human FANCJ protein sequence, two FANCJ homologs could be identified in the Arabidopsis genome, named FANCJA (At1g20750) and FANCJB (At1g20720). The AtFANCJ proteins are 27.5% and 27.4% identical to HsFANCJ, respectively and 66.2% identical to each other. AtFANCJA comprises of 5697 bp with 25 exons and 24 introns, while AtFANCJB harbours 30 exons and 29 introns in 7137 bp (Fig 1). Only two genes on chromosome 1 thereby separate the genes from each other. We analysed expression profiles for both genes based on the TraVA database . Thereby FANCJA exhibited an extremely low expression, with the highest expression level found in anthers. However, the expression level of FANCJB was at least one order of magnitude higher, with the highest expression found in meristems. For the functional characterization of these FANCJ homologs in Arabidopsis, one T-DNA insertion line for each gene was analysed. In fancja-1 (SALK_079991) the T-DNA insertion is located in intron 24, while the T-DNA in fancjb-1 (SALK_101493) is inserted in intron 10 and associated with a genomic deletion of 15 bp (S1 Fig). Additionally, we generated further mutants for FANCJA and FANCJB via Cas9-mediated mutagenesis. The mutagenesis of AtFANCJA was performed using a double nickase approach with S. pyogenes Cas9, as previously described . Therefore, protospacers in intron 16 (5’-GAATAAGAATGCATCAGTTT-3’) and spanning intron 16/exon 17 (5’-AGAACCAAGAGGAGGATCCA-3’) were used. With this approach, two additional fancja mutant lines could be generated, termed fancja-2 and fancja-3 (S2 Fig). For the mutagenesis of AtFANCJB, two independent S. aureus Cas9 nuclease approaches were performed . The target sequences were located spanning exon 9/intron 10 (5’-TTACCTCTACTACTTCACAC-3’) and spanning intron 15/exon 16 (5’-TATCTTTGTTCCAGGTGTGG-3’). With each approach, one mutant line was obtained and named fancjb-2 and fancjb-3, respectively (S4 Fig). The mutations in all produced lines lead to frameshifts within the open reading frame of the corresponding gene and thereby generated premature stop codons, most likely leading to an abortion of translation (S3 and S4 Figs). The fancja and fancjb mutant lines were indistinguishable from WT plants with regards to their growth phenotype, and were fully fertile (S5 Fig).
AtFANCJA is located at the locus At1g20750 and comprises of 25 exons (red boxes) and 24 introns (black bars) in 5697 bp. The T-DNA insertion in fancja-1 is integrated in intron 24. The mutations in fancja-2/3 are located spanning intron 16/exon 17, the insertions in fancja-4/5 are inserted in exon 17. AtFANCJB (At1g20720) consists of 7137 bp in 30 exons (green boxes) and 29 introns (black bars). In fancjb-1, a T-DNA is inserted in intron 10, fancjb-2 harbours a deletion in exon 16 and fancjb-3 contains a deletion in exon 9.
As human FANCJ is a known factor involved in the FA pathway, we were interested in the function of the Arabidopsis FANCJ homologs in ICL repair. To test this, we analysed the obtained fancja and fancjb T-DNA and CRISPR/Cas9 mutant lines on their sensitivity towards the ICL inducing agent Mitomycin C (MMC). Therefore, we treated one-week-old plantlets of the individual lines with different MMC concentrations, incubated them for an additional two weeks and measured the resulting fresh weight. The fresh weight of treated plants was normalized to the fresh weight of untreated plants of the same line, to exclude the influence of growth differences. As a control, wild-type (WT) plants were treated accordingly. For all three tested fancja mutant lines, no significant differences in fresh weight compared to WT plants could be observed following MMC treatment (Fig 2A). In contrast to this, the three fancjb mutant lines exhibited a comparable hypersensitivity after treatment with 5, 10 and 15 μg/ml MMC, resulting in a significantly reduced fresh weight compared to WT plants (Fig 2B). This indicates an involvement of AtFANCJB in ICL repair. As mutants from other helicases such as AtRECQ4A, AtFANCM and AtRTEL1 show sensitivity in addition to a hyperrecombination phenotype, we were wondering whether the same holds true for AtFANCJB. Therefore, we crossed the fancjb-2 and fancjb-3 mutant lines with the IC9C reporter line . This line harbours an interrupted GUS gene that enables the quantification of interchromosomal recombination events upon restoration. However, both fancjb mutants tested did not differ in their recombination frequencies from that of WT plants (Fig 2D). Thus, AtFANCJB is not involved in the suppression of HR in plants.
The fresh weight of the individual lines in response to MMC treatment was determined in relation to untreated control plants. Five independent assays were performed and mean values with standard deviations (error bars) were calculated. (A) The relative fresh weight of all fancja mutant lines did not differ significantly from WT plants. (B) All three fancjb mutant lines depicted a significantly reduced fresh weight compared to WT plants at 5, 10 and 15 μg/ml MMC concentrations. (C) Both fancja fancjb double mutant lines were hypersensitive against MMC treatment, on a level comparable to the fancjb single mutant. (D) Homologous recombination frequency was determined with the IC9C reporter in three independent assays and mean values with standard deviations (error bars) were calculated. The number of blue sectors per plant in both fancjb mutant lines did not differ from WT plants. Statistical differences were calculated using the two-tailed t-test with unequal variances: * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant.
AtFANCJA is not involved in ICL repair
Although no direct function for FANCJA in ICL repair could be found, the homolog could still perform a possible hidden role, with a backup function for FANCJB. To address this question, we aimed to analyse fancja fancjb double mutants. As the two genes are both located on chromosome 1, separated by only 10 kb, the utilisation of crossbreeding for double mutant generation was not possible. To circumvent this problem, we performed a Cas9-mediated mutagenesis of FANCJA in the fancjb-1 mutant line. Therefore, we used the same double nickase approach as for the fancja single mutant generation. Two individual mutant lines for FANCJA could be obtained in the fancjb-1 mutant background, resulting in the double mutants fancja-4 fancjb-1 and fancja-5 fancjb-1. The FANCJA mutations in both double mutants lead to a frameshift in the open reading frame of the FANCJA gene, thus resulting in a premature stop codon (S3 Fig). We tested the obtained double mutants on their sensitivity towards MMC in comparison to the fancja-1 and fancjb-1 single mutants and WT plants. While fancja-1 mutants did not display MMC hypersensitivity, fancjb-1 mutants showed a significantly reduced fresh weight in comparison to the WT (Fig 2C). Interestingly, both fancja fancjb double mutant lines exhibited hypersensitivity towards MMC on a level comparable to the fancjb-1 single mutant, in all tested concentrations. Thus, AtFANCJA does not contribute to ICL repair and AtFANCJB seems to be the only functional FANCJ homolog in Arabidopsis.
AtFANCJB acts in a common ICL repair pathway with the nuclease AtFAN1 and the helicase AtRECQ4A
As we were able to demonstrate a role for AtFANCJB in ICL repair, we wanted to define in detail as to which branch of the quite complex ICL repair network of plants it is acting in. Therefore, we generated double mutants with known ICL repair factors, by crossbreeding of the corresponding single mutants with fancjb-1, and analysed them in sensitivity assays with MMC. The nuclease FAN1 belongs to a group of FA-associated factors that are necessary for ICL repair in humans, but not causal for FA upon mutation [5,45]. FAN1 was postulated to be involved in the initial unhooking step in ICL repair, where the nuclease likely cuts opposite from either MUS81-EME1 or XPF/ERCC1 . Interestingly, FAN1 is not conserved in all eukaryotes, as functional homologs could be found in mammals and S. pombe, whilst Xenopus FAN1 does not seem to be involved in ICL repair and no homologs were found in Drosophila or S. cerevisiae [47,48]. For AtFAN1, a conserved function in ICL repair upstream of both the RecQ helicase RECQ4A and the ATPase RAD5A, but in parallel to the nuclease MUS81, could be demonstrated . We were therefore now interested as to whether AtFANCJB, as functional homolog of a known FA factor, acts together with the FA-associated nuclease AtFAN1 in ICL repair. Thus, we tested the fancjb-1 fan1-1 double mutant for its sensitivity against MMC (Fig 3A). Both fancjb-1 and fan1-1 single mutants exhibited a comparable hypersensitivity against all tested concentrations of MMC, reflecting the roles of FANCJB and FAN1 in ICL repair. The double mutant featured a reduced fresh weight compared to WT plants, but did not differ significantly from either single mutant. Thus, FANCJB and FAN1 seem to participate in a common pathway in ICL repair.
Relative fresh weight of double mutants, the respective single mutants and wild-type plants (WT) was determined after MMC treatment. Independent assays were performed at least 4 times and mean values with standard deviations (error bars) were calculated. (A) The relative fresh weight of fancjb-1 fan1-1 double mutants was comparable to both single mutant lines in all tested MMC concentrations. (B) The fancjb-1 recq4A-4 double mutants exhibited a relative fresh weight that did not significantly differ from fancjb-1 single mutants. Statistical differences were calculated using the two-tailed t-test with unequal variances: * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant.
The RecQ helicase AtRECQ4A is the functional homolog of the human BLM helicase and possesses important functions in various DNA repair and recombination processes [33,49]. Although recq4a mutants in Arabidopsis do not exhibit strong MMC sensitivity, a role in ICL repair could be unveiled by double mutant analyses. Thereby, RECQ4A could be classified into a repair pathway parallel to the FA helicase FANCM . Consequently, it was of special interest to classify the other FA helicase from Arabidopsis, FANCJB, into the ICL repair pathways with regard to its cooperation with the RecQ helicase RECQ4A. While the recq4A-4 single mutant only showed a slightly reduced fresh weight compared to WT plants following MMC treatment with 5 and 10 μg/ml, the fancjb-1 recq4A-4 double mutant was comparable to the fancjb-1 single mutant in all tested MMC concentrations (Fig 3B). This hints to a joint involvement of both helicases in ICL repair.
AtFANCJB cooperates with the PRR factors AtRAD5A and AtREV3 in ICL repair
AtRAD5A was shown to be the functional homolog of the PRR factor ScRad5 and acts in the repair of DNA methylations and crosslinks . Thereby, a function independent of NER, SSB repair and MMEJ could be demonstrated . In ICL repair, RAD5A mediates a RECQ4A-independent sub-pathway downstream of the FA-associated nuclease FAN1 . In the two-branched model of PRR in yeast, Rad5 is involved in the error-free pathway, both in the polyubiquitination of PCNA and mechanistically as a DNA translocase [50–52]. The error-prone pathway is mediated by translesion synthesis (TLS), where specialised polymerases are able to bypass DNA lesions . REV3 is the catalytic subunit of the TLS polymerase Zeta, in which for the Arabidopsis homolog, an involvement in the repair of different DNA damages including ICLs was shown [40,54,55]. Furthermore, the parallel involvement of REV3 and RAD5A in DNA repair demonstrated the conservation of the two-branched PRR model in Arabidopsis . For further analysis of the role of FANCJB in ICL repair, we analysed double mutants with the PRR factors RAD5A and REV3 in response to MMC (Fig 4). Atrad5A-2 mutants exhibited a strong MMC sensitivity in all tested concentrations, while fancjb-1 mutants depicted hypersensitivity after treatment with only 5 and 15 μg/ml (Fig 4A). For fancjb-1 rad5A-2 double mutants, no further reduction in fresh weight could be detected, resulting in a fresh weight comparable to rad5A-2 single mutants. For rev3-5 mutants, hypersensitivity against all tested MMC concentrations could be observed (Fig 4B). The simultaneous mutation of FANCJB and REV3 in the corresponding double mutant resulted in MMC hypersensitivity on a level comparable to the rev3-5 single mutant in all tested concentrations. These results imply a cooperation of FANCJB with both RAD5A and REV3 in ICL repair, combining the error-free and error-prone branch of PRR into a common pathway with a FA factor in Arabidopsis.
Relative fresh weight of double mutants in comparison to the corresponding single mutants and wild-type plants (WT) was determined after MMC treatment. At least 4 independent assays were performed and mean values with standard deviations (error bars) were calculated. (A) The relative fresh weight of fancjb-1 rad5A-2 double mutants was comparable to the rad5A-2 single mutant in all tested MMC concentrations. (B) The fancjb-1 rev3-5 double mutant exhibited a relative fresh weight that was not significantly different from the rev3-5 single mutant after treatment with MMC. (C) In fancjb-1 mus81-1 double mutants, a significantly reduced fresh weight compared to both single mutants could be observed after 5 and 10 μg/ml MMC treatment. Statistical differences were calculated using the two-tailed t-test with unequal variances: * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant.
AtMUS81 and AtFANCJB act independently in ICL repair
The endonuclease AtMUS81 was shown to act on various branched DNA structures together with its complex partner EME1 and is thereby involved in the repair of a variety of DNA lesions [38,57]. AtMUS81 defines a special kind of backup pathway in ICL repair, where the nuclease acts separate from most other known factors [19,31]. For mammalian MUS81, an involvement parallel to FA signalling was demonstrated in ICL repair . Interestingly, the simultaneous mutation of MUS81 and the FA helicase FANCM in Arabidopsis leads to severe developmental defects, indicating independent roles in the repair of replicative DNA damage [18,20]. These findings raised questions of the functional relationship of MUS81 and FANCJB in ICL repair. Therefore, we analysed fancjb-1 mus81-1 double mutants on their sensitivity towards MMC, as no growth defects were detectable in untreated plants. Both the fancjb-1 and mus81-1 single mutants exhibit a reduced fresh weight after treatment with 5, 10 and 15 μg/ml MMC, compared to WT plants (Fig 4C). In fancjb-1 mus81-1 double mutants, an additive sensitivity could be observed at 5 and 10 μg/ml MMC. This led us to the conclusion that MUS81 mediates an independent pathway from FANCJB in ICL repair.
The helicases AtRTEL1 and AtFANCJB are independently involved in ICL repair and replication-associated DNA repair in the root meristem
FANCJ and RTEL1 are closely related helicases that both belong to the XPD helicase family . This helicase family encompasses a number of conserved proteins important for genome stability, and FANCJ and RTEL1 homologs are both present in animals and plants [21,60]. For mammalian RTEL1, important functions in telomere stability have been demonstrated, and RTEL1 was shown to be necessary for T-Loop dismantling during replication [22,27]. RTEL1 homologs exhibit important roles in ICL repair, as shown for C. elegans and human cell lines . For Arabidopsis, a conserved function for RTEL1 in ICL repair could be demonstrated, where the helicase interestingly acts in a common pathway with the endonuclease MUS81, but in a pathway parallel to the helicases FANCM and RECQ4A . Of course, this raised questions regarding the genetic interaction of RTEL1 and FANCJB in ICL repair. Therefore, we analysed fancjb-1 rtel1-1 double mutants in sensitivity assays with MMC treatment (Fig 5A). For rtel1-1 single mutants, only a slight hypersensitivity could be observed after treatment with 5 μg/ml MMC, while fancjb-1 single mutants exhibited a strong reduction of fresh weight compared to WT plants at 5, 10 and 15 μg/ml MMC concentrations. In fancjb-1 rtel1-1 double mutants a further reduction of fresh weight, compared to both single mutants, could be observed after treatment with 10 and 15 μg/ml MMC. Thus, FANCJB and RTEL1 are involved in independent pathways of ICL repair.
(A) Relative fresh weight of fancjb-1 rtel1-1 double mutants, the respective single mutants and wild-type plants (WT) was determined after MMC treatment (n = 3). The fancjb-1 rtel1-1 double mutant depicted a significantly reduced fresh weight compared to both single mutants after treatment with 10 and 15 μg/ml MMC. (B) Root length of 10-day-old fancjb-1 rtel1-1 double mutants was determined in comparison to the respective single mutants and WT plants (n = 4). The roots of fancjb-1 rtel1-1 double mutants were significantly shorter than roots of either single mutant. (C) Cell death analysis in the root meristem of fancjb-1 rtel1-1 double mutants in comparison to the corresponding single mutants and WT plants (n = 5, scale bar = 50 μm). In fancjb-1 rtel1-1 double mutants, a significantly increased amount of dead transiently amplifying (TA) cells, in comparison to both single mutants, could be observed. Statistical differences were calculated using the two-tailed t-test with unequal variances: * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant.
In Arabidopsis, RTEL1 was furthermore shown to be important for correct replication, as mutants depict a slow growth phenotype and shortened roots [21,29]. Thereby, a role for RTEL1 in the repair of replication-associated DNA damage in parallel to FANCM, MUS81 and RECQ4A, was demonstrated. As FANCJB is closely related to RTEL1, we were now interested in the function of both factors in replication-associated DNA repair. Spontaneously occurring DNA damage can lead to blockades of the replication machinery, therefore slowing cell division. As root growth in plants relies on cell divisions within the root meristem, it is a suitable system for documenting the impact of replication-associated DNA damage . Therefore, we analysed the root length of 10-day-old fancjb-1 rtel1-1 double mutants in comparison to the corresponding single mutants and WT plants (Fig 5B). While both single mutant lines exhibited a root length comparable to WT plants under these conditions, root length in the double mutant was significantly reduced, with a remaining length of less than a third of WT roots. We wanted to take a closer look at the roots and the possible cause of this phenotype, therefore we analysed cell death in the root meristem. This is based on the staining of roots from five-day-old seedlings with the nucleic acid intercalating dye, propidium iodide (PI). PI is only able to pass the perforated cell membrane of dead cells and thereby allows the differentiation between living and dead cells. The stem cell niche of the root meristem consists of stem cells that are grouped around the quiescent centre. Via the constant division of these cells, the different cell types in roots are formed. The transiently amplifying (TA) cells are located apical of the stem cell niche, that are characterized by frequent cell divisions and therefore are especially well suited for the analysis of replication-associated DNA repair . Cells of the root meristem are extraordinarily sensitive against genotoxic stress and feature a selective occurrence of programmed cell death, even in response to a low level of DNA damage . We analysed the number of dead TA cells in root tips of fancjb-1 rtel1-1 double mutants in comparison to both single mutants and WT plants (Fig 5C). No dead TA cells were visible in WT roots and both single mutants did not depict a significantly different number of dead TA cells compared to WT roots. Consistent with the root length results, in fancjb-1 rtel1-1 double mutants, an increased number of dead TA cells per root could be observed. Thus, the short root phenotype of fancjb-1 rtel1-1 double mutants seems to be caused by an increased cell death in the root meristem. This is likely because of parallel functions of both helicases in the repair of replication-associated DNA damage.
AtRTEL1 and AtFANCJB act in parallel pathways in the maintenance of rDNA repeat stability
RTEL1 was shown to be an important factor for the stability of repetitive DNA sequences. The 45S rDNA loci on chromosome 2 and 4 build a large cluster of repetitive sequences in the Arabidopsis genome. In a previous study, we were able to demonstrate an important function for AtRTEL1 in stabilizing 45S rDNA repeats. 45S rDNA repeat number in rtel1-1 mutants is reduced to about half of that of WT plants. Thereby, a parallel involvement of RTEL1 and the RTR complex partner RMI2 in 45S rDNA maintenance could be observed, as double mutants suffer from a further diminished 45S rDNA copy number to only a third of WT plants . Human FANCJ was postulated to act in supressing microsatellite instability, in a function independent of the FA pathway [63,64]. This led us to question whether AtFANCJB might also be involved in rDNA maintenance. To determine this, we analysed the relative number of 45S and 5S rDNA repeats in two-week-old fancjb-1 single and fancjb-1 rtel1-1 double mutants (third homozygous generation), compared to rtel1-1 mutants and WT plants, via quantitative real-time PCR (Fig 6A). We were able to reproduce the strong reduction of 18S, 5.8S and 25S rDNA amounts in rtel1-1 single mutants, while no reduction in rDNA copy number could be observed in fancjb-1 single mutants compared to WT plants. Astonishingly, in fancjb-1 rtel1-1 double mutants, the amount of all three 45S rDNA coding sequences was significantly further reduced, compared to rtel1-1 single mutants, to only about 30% of WT plants. Additionally, we included an analysis of 5S rDNA repeat number. The 5S rDNA loci, coding for the 5S rRNA, are located in the pericentromeric regions of chromosome 3, 4 and 5 in Arabidopsis [65,66]. Interestingly, 5S rDNA copy number in the rtel1-1 single mutant did not differ from WT copy number and fancjb-1 single mutants even depicted a slight increase in the amount of 5S rDNA. In contrast to this, the fancjb-1 rtel1-1 double mutant possessed a significantly reduced copy number of about 80% of WT plants. To confirm the results concerning the 45S rDNA with a cytological method, visualizing the reduction in rDNA copy number, we analysed the fancjb-1 rtel1-1 double mutant, the corresponding single mutants and WT plants in a FISH (fluorescence in situ hybridisation) analysis (Fig 6B and 6C). Therefore, we used a 45S rDNA probe labelled with Atto488 and DAPI staining in chromatin spreads, as described before . With this method, we were able to confirm the drastic reduction in 45S rDNA amount in fancjb-1 rtel1-1 double mutants compared to both single mutant lines. These results indicate an important function for FANCJB in the maintenance of the 45S rDNA repeats in parallel to RTEL1. Furthermore, both helicases are likely to also be involved in the stabilisation of 5S rDNA repeats. This further underlines a key function for FANCJB in maintaining genome stability in Arabidopsis, not only in ICL repair, but also in the homeostasis of repetitive sequences.
(A) The rDNA repeat amount was determined by quantitative real-time PCR in the fancjb-1 rtel1-1 double mutant and corresponding single mutants, in relation to WT plants (n = 5). The 45S rDNA repeat number was determined using individual primer pairs for the 18S, 5.8S and 25S rDNA sequences. While the 45S rDNA copy number in the fancjb-1 single mutant was comparable to WT plants, the fancjb-1 rtel1-1 double mutant exhibited a copy number significantly reduced to both single mutants. The 5S rDNA repeat number also showed a significant reduction in the fancjb-1 rtel1-1 double mutant compared to both single mutants. (B) The reduction in 45S rDNA amount was additionally quantified by FISH (fluorescence in situ hybridization), using an Atto488 labelled 45S rDNA probe and DAPI staining of chromatin (n = 4). The area of the 45S rDNA was significantly reduced in the fancjb-1 rtel1-1 double mutant compared to both single mutants. (C) Exemplarily chosen nuclei from each genotype depicting chromatin (blue) with the four 45S rDNA loci (green, scale bar = 10 μm). Statistical differences were calculated using the two-tailed t-test with unequal variances: * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant.
The human FANCJ helicase is involved in the Fanconi Anemia (FA) pathway of ICL repair and mutations in the corresponding gene lead to the eponymous hereditary disease FA [2–4]. In this study, we were able to identify FANCJB from Arabidopsis thaliana as the functional homolog of FANCJ in ICL repair. Furthermore, we were able to classify FANCJB into the ICL repair pathways of Arabidopsis and found a surprising involvement of FANCJB in the maintenance of rDNA repeat number in parallel to the related helicase RTEL1.
FANCJB is the functional Arabidopsis FANCJ homolog in ICL repair
With bioinformatical analyses, we were able to identify two FANCJ homologs in the Arabidopsis thaliana genome, FANCJA and FANCJB. While both proteins share a high identity in amino acid composition, we could demonstrate a functional divergence. FANCJB is involved in ICL repair, apparent by the hypersensitivity of three independent fancjb mutant lines against MMC treatment. This is in accordance with the function of human FANCJ in the FA pathway of ICL repair, but also for FANCJ homologs in other animal systems [2–4,67,68]. As no such role was apparent for AtFANCJA, we further investigated a possible function of this homolog. The power of the CRISPR/Cas9 technology enabled us to generate double mutants . A genetic approach for combining both mutant alleles was excluded as the genes are separated by only 10 kb on chromosome 1. Upon analysing the double mutant, we did not find any backup role for FANCJA in ICL repair. Thus, the function of FANCJA still remains elusive. It is noticeable that the expression level of FANCJA is at least one order of magnitude lower than the expression of FANCJB, and in general is hardly detectable . This led us to the conclusion that FANCJB is the only functional Arabidopsis FANCJ homolog in ICL repair. As we were able to characterize a FANCJ homolog in a non-animal system for the first time, our results demonstrate that the role of FANCJ seems to be evolutionary conserved between animals and plants.
Defining the role of FANCJB in the ICL repair pathway network of Arabidopsis
As we were able to identify FANCJB as functional FANCJ homolog in ICL repair, we were further interested in defining the role of FANCJB within the different repair pathways of Arabidopsis. Past analyses from our group defined a complex ICL repair network, with the already characterized factors FAN1, RECQ4A, RAD5A, REV3, MUS81 and RTEL1. With double mutant analyses, we now wanted to clarify if FANCJB acts in common or separate pathways with these proteins.
The nuclease FAN1 is a FA-associated factor that is essential for ICL repair in humans . For the Arabidopsis homolog, an involvement in ICL repair has been demonstrated, where the nuclease acts upstream of two sub-pathways mediated by the RecQ helicase RECQ4A and the PRR ATPase RAD5A . Our results now indicate a common involvement of FAN1 with the FA helicase FANCJB. As FAN1 is not conserved in all eukaryotes, its involvement in ICL repair seems to be also diverse in the different organisms where it is present. For chicken DT40 cells, a synergistically increased number of MMC-induced chromosomal aberrations could be determined in cells lacking both FAN1 and FANCJ . Thus, there are clear differences in animals and plants in whether or not these two proteins act in a common repair pathway. It is tempting to speculate that in Arabidopsis both proteins cooperate with their complementing biochemical activities to process cross-linked DNA, to set it up for downstream repair. The nuclease might be required for breaking the DNA close to the CL, whereas the helicase might use the broken end to unwind the DNA at the damaged site.
AtRECQ4A is the functional homolog of the human BLM and the yeast Sgs1 helicase [33,49]. Studies from our group identified RECQ4A as crucial CL repair factor, defining a parallel pathway to RAD5A and MUS81 . We were now able to show a common involvement of RECQ4A and FANCJB in ICL repair. For human FANCJ and BLM, a direct interaction could be demonstrated and the cellular level of BLM was shown to be dependent on FANCJ. Furthermore, BLM and FANCJ partially co-localize after MMC treatment of human cells . This is in accordance with an involvement of BLM in the FA pathway, where additionally a function of BLM in the activation of FANCD2 was revealed . Thus, the cooperation of both helicases in a common pathway seems to be evolutionary conserved. After the initial unwinding of the damaged DNA by FANCJB, RECQ4A might be involved in the further processing of these intermediates. These intermediates might then be processed by homologous recombination.
Post-replicative repair (PRR) is an important mechanism for the bypass of a wide variety of replication-blocking DNA damages, including ICLs. Thereby, PRR splits into an error-free and an error-prone branch, dependent on the ubiquitination state of PCNA. In yeast, the E3 ubiquitin ligase Rad5 was identified as an important positive regulator of the error-free PRR branch, with an additional mechanistical function as DNA translocase . The error-prone pathway relies on the function of TLS polymerases that mediate replication past lesions, thereby potentially inducing mutations due to the lacking proofreading activity . The two-branched PRR model is represented in Arabidopsis thaliana by the Rad5 homolog RAD5A and the TLS polymerase subunit REV3, that are both involved in ICL repair [35,40]. Our findings now suggest a common involvement of both PRR factors, together with FANCJB, in ICL repair, possibly with FANCJB acting upstream of the two separate PRR pathways. For Mph1, the yeast FANCM homolog, an epistatic interaction with Rad5-mediated DNA damage bypass was postulated . This is especially interesting as the FA-pathway is not conserved in yeast, similar to Arabidopsis. In contrast to this, the human Rad5 homolog HLTF counteracts FANCJ in the replication stress response, indicating different circumstances in a situation with the presence of the FA pathway . Also the interaction of error-prone PRR and FA-factors differs between organisms, as a common involvement of REV3 and FANCC in CL repair in DT40 cells was demonstrated, while human FANCJ enhances TLS after ICL induction [74,75]. For Arabidopsis, it seems that after the initial processing of the CL intermediates by FAN1 and FANCJB, in a sub-pathway, post-replicative repair factors can take over the processing of these intermediates as an alternative to the processing by RECQ4A.
FANCJ is ranked in a subclass of Fe-S helicases, including the helicases XPD, CHL1 and RTEL1 . The involvement of AtRTEL1 in ICL repair was demonstrated before and the genetic interaction of the closely related helicase with FANCJB was of central interest in our study . As rtel1 mutants only depict a minor hypersensitivity after MMC treatment, FANCJB seems to fulfil a more important function in ICL repair. Nevertheless, as we found a synergistic hypersensitivity in fancjb rtel1 double mutants, this implies independent roles for both helicases.
We further analysed the genetic interaction of FANCJB with the endonuclease MUS81. In Arabidopsis, MUS81 was postulated to define an important backup pathway for maintaining genome stability, as double mutants with a wide variety of DNA repair factors exhibit increased genotoxin sensitivity, severe growth retardation or synthetic lethality [31,37–39]. Our results clearly show that FANCJB is not processing CLs in a common pathway with MUS81 in plants. For mammalian systems, MUS81 was also demonstrated to contribute to genome stability, independently of the FA pathway. FANCD2 monoubiquitination following ICL induction was shown to be independent of MUS81, and ICL repair relies on the action of both FA-factors and MUS81 [58,76]. So in principle, two independent ways to initiate CL repair exist in plants .
With these analyses, we were now able to include FANCJB into the ICL repair network of Arabidopsis thaliana (Fig 7). FANCJB and FAN1 act in a common pathway initiating the repair reaction. The resulting intermediates are then alternatively processed by three sub-branches, defined by the RecQ helicase RECQ4A, in parallel to both PRR factors REV3 and RAD5A. A pathway separate from FANCJB is mediated by the backup-endonuclease MUS81. In this ICL repair pathway, the helicase RTEL1, for which we could now also show independent functions from FANCJB, is involved. Obviously both independent pathways require a Fe-S cluster kind of helicase for unwinding complex CL-associated DNA structures.
FANCJB acts together with FAN1 in interstrand CL (ICL) repair, most likely upstream of three subpathways defined by RECQ4A, REV3 and RAD5A. FANCJB and MUS81 act in independent pathways, whereby RTEL1 could be classified into a common pathway with MUS81, separate from FANCJB.
FANCJB and RTEL1 are independently important for the stabilization of rDNA repeat number
As both XPD-like helicases FANCJB and RTEL1 are present in Arabidopsis, and double mutants depict a remarkably viable phenotype, this not only enabled analyses of both factors in ICL repair, but also concerning further functions of the helicases in maintaining genome stability. Double mutants for rtel1 and the C. elegans FANCJ homolog dog-1 depict a synthetically lethal phenotype, emphasizing the essential roles of both factors . For AtRTEL1, important functions in replication-associated DNA repair have been demonstrated, as mutants exhibit various cell proliferation defects including a prolonged S-phase . Furthermore, an increased cell death in the root meristem of rtel1 single mutants, and an even further enhanced effect in double mutants with various DNA repair factors, underlined the significance of this helicase for correct DNA replication [21,30]. Our results also indicate a function for FANCJB in replication-associated DNA repair, as double mutants with rtel1 exhibited a drastic shortening of roots, accompanied with an increase of cell death in the root meristem compared to both single mutants. Thus, FANCJB and RTEL1 independently contribute to a correct replication in the root meristem.
RTEL1 was demonstrated to be involved in the maintenance of repetitive DNA sequences. Recently, an important role for AtRTEL1 in the processing of telomeric recombination intermediates, arising through a RAD51-dependent HR pathway in the absence of telomerase, has been demonstrated . Interestingly, AtRTEL1 was also shown to be involved in the maintenance of 45S rDNA repeat stability. The 45S rDNA genes are arranged in tandem repeats on chromosomes 2 and 4 and form one of the largest repetitive elements in the Arabidopsis genome [78,79]. The stability of the rDNA gene clusters is of special importance as they code for the catalytic rRNA core of ribosomes, essential for protein biosynthesis in all organisms. Our group demonstrated that the mutation of RTEL1 in Arabidopsis plants is associated with a dramatic loss of 45S rDNA repeats to only 50% of WT copies. Thereby, RTEL1 acts independently of the RTR complex partner RMI2, as double mutants depict an even further loss of 45S rDNA copies . This essential role for RTEL1 has been recently confirmed in the moss Physcomitrella patens . Our analysis now also indicates a role for FANCJB in rDNA maintenance, as double mutants with RTEL1 exhibit a drastic loss of 45S rDNA repeat number compared to either single mutant, resulting in a copy number of only 30% of WT plants. We extended our analysis to include the investigation of 5S rDNA repeat number. In Arabidopsis thaliana, the 5S rDNA repeats are physically separated from the 45S rDNA loci and are located at the pericentromeric regions of chromosomes 3, 4 and 5. In our analysis, the 5S rDNA repeats seem to be more stable, as the loss of either RTEL1 or FANCJB did not result in a decrease in copy number, only the joint mutation of both helicases leads to a reduced 5S rDNA repeat number to about 80%. This indicates differences for the stability of 5S and 45S rDNA loci in Arabidopsis, and further analyses of factors differentially involved in the stabilisation might shed light on the underlying mechanism. Previous studies support this finding, as the copy number variation of 45S and 5S rDNA repeats in different Arabidopsis ecotypes did not show any correlation . Thus, the epigenome might play an important role as transcription of the two rDNA species is mediated by different RNA polymerases, and factors associated with chromatin were shown to influence 45S, but not 5S, rDNA repeat stability [82,83]. As a role in the maintenance of epigenetic stability at G4-prone DNA sequences was shown in animals for FANCJ, it is tempting to speculate on such a possible function in Arabidopsis as well . A further factor might be the localization on different genomic loci, as the 5S and 45S rDNA repeats in P. patens are linked in a common arrangement and were shown to react with a similar copy number reduction after RTEL1 mutation . As we now uncovered this change in rDNA copy number, it would also be interesting to analyse these effects in a multi-dimensional approach in the future, analysing the variability between single plants, at different developmental stages and in subsequent generations, as opposed to only a pool of plants from a single mutant generation. Recently, the mechanism of rDNA repeat stabilisation during meiosis was revealed, as HR is specifically downregulated in the nucleolus and DNA breaks are repaired via NHEJ . It would be of special interest to see whether there is an effect on rDNA repeat stability following the depletion of FANCJB and RTEL1, during meiosis.
When we originally discovered rDNA instability in the rtel1 rmi2 double mutant background, we postulated that this phenotype goes along with a general instability of repeated sequences . This was because both single, as well as the double mutant, showed a hyperrecombination phenotype for a repeated transgene marker. However, this is not the case for AtFANCJB, which as demonstrated by the use of the same marker (Fig 2D), is not involved in the suppression of homologous recombination. Therefore, we postulate that the helicases are required for the processing of complex replication intermediates. Due to its repetitive nature and sequence composition, the ribosomal DNA is especially prone to the formation of such complex secondary structures. G-quadruplexes (G4) are extremely stable secondary structures stabilized by Hoogsteen hydrogen bonding of guanine bases [86,87]. Analyses from P. patens also demonstrated the presence of a highly G4-prone sequence in the linker between 5S and 45S rDNA repeats . For both FANCJ and RTEL1 helicases, functions in the unwinding of G4 DNA structures have been postulated, hinting now to a link between rDNA repeat stability and G4 DNA unwinding in Arabidopsis. The analyses of RTEL1 in G4 homeostasis mainly concentrated on telomeres where mammalian RTEL1 was postulated to unwind telomeric G4 structures [22,88]. But also a function for human RTEL1 in the suppression of trinucleotide repeat expansions, probably by the unwinding of hairpin structures, was postulated, allowing the possibility of a broader substrate spectrum . FANCJ and its homologs from various organisms have been thoroughly described in respect to their role in G4 metabolism . The ability of human FANCJ to unwind G4 DNA structures was shown to be independent of the FA pathway and an in vivo function could be demonstrated, as FANCJ depleted cell lines depicted hypersensitivity towards G4 ligands . The identification of DOG-1 in C. elegans was even based on a mutant phenotype with deletions in genes containing polyguanine tracts, now known to be based on the function of the helicase in unwinding G4s [90,91]. Similar phenotypes were also reported for human cell lines, furthermore a function for mammalian FANCJ in the maintenance of microsatellites was also demonstrated [63,64,92]. For the human RTR complex helicase BLM, an G4 unwinding function in vitro has also been shown and recent studies hint to an in vivo role in the stabilisation of G4-rich sequences [93,94]. Based on the knowledge about the helicases FANCJ and RTEL1, as well as the RTR complex, it is now tempting to speculate if the effects of these factors we see on rDNA repeat stability in Arabidopsis is based on their function to unwind G4 structures. Interestingly, it was shown before that the non-transcribed spacer (NTS) sequence, naturally located between rDNA genes, enhances homologous recombination when positioned next to a recombination substrate . This might be based on the specific blockage of the replication fork at a possible internal replication fork barrier (RFB). It could be that in the absence of FANCJB, a barrier for replication forks on G4-prone rDNA sequences is similarly induced, thereby increasing recombination at these sites, leading to a loss of rDNA copies, but not detectable in our artificial recombination marker lacking such G4-rich regions. It would be interesting to test this hypothesis by additional analyses with a modified reporter system. The helicases FANCJB and RTEL1 might complement each other in the unwinding of G4 DNA, but in the absence of both, the presence of persisting G4 structures might strongly hinder replication, leading to a dramatic loss of still non-replicated rDNA copies. As the absence of either helicase in humans is associated with a hereditary disease, Fanconi Anemia and Hoyeraal-Hreidarsson syndrome, respectively, it is of utmost interest to see if rDNA instability occurs in humans and has pathological consequences.
Materials and methods
Plant material and growth conditions
The Arabidopsis thaliana lines used in this study were in the Columbia (Col-0) background. For the characterization of FANCJA and FANCJB in Arabidopsis, the T-DNA insertion lines fancja-1 (SALK_079991) and fancjb-1 (SALK_101493), respectively, were used. Additional fancja mutant lines were generated using CRISPR/Cas9 mediated mutagenesis with a S. pyogenes Cas9 double nickase approach, as described before . For the generation of fancja single mutants, mutagenesis was performed in Arabidopsis wild type plants, for fancja fancjb double mutants, mutagenesis was carried out in fancjb-1 mutants. The fancjb-2 and fancjb-3 mutants were produced by S. aureus Cas9-mediated mutagenesis, as described in . Further double mutants were generated by crossbreeding of fancjb-1 with the previously described fan1-1 (GABI_815C08), recq4A-4 (GABI_203C07), mus81-1 (GABI_113F11), rad5A-2 (SALK_047150), rev3-5 (SALK_126789) and rtel1-1 (SALK_113285) mutant lines from the SALK and GABI-Kat collections, respectively [19, 21, 33, 35, 38, 40, 96, 97]. For cultivation in the greenhouse, plants were sown on a 1:1 mixture of Floraton 3 (Floragard, Oldenburg, Germany) and vermiculite (2–3 mm, Deutsche Vermiculite Dämmstoff, Sprockhövel, Germany) and grown at 22 °C with 16 h light and 8 h dark. The cultivation of plants for assays was performed under axenic conditions as described before .
To analyse fertility, Arabidopsis plants were grown in the greenhouse. Five mature siliques of five plants per line were harvested and transferred into 70% EtOH. After incubation overnight, the silique length and number of seeds per silique were determined using a binocular microscope.
Sensitivity analysis was performed as previously described . Plants were grown in axenic culture for 7 days, and then 10 seedlings were transferred into each well of a six-well plate containing 5 ml GM medium for negative controls, or 4 ml for genotoxin treated samples. The following day, 1 ml genotoxin solution was added to obtain the intended concentration. After 13 days of incubation, the fresh weight of the plants was determined. The fresh weight of each treated sample was normalised to the fresh weight of the untreated control.
HR assays utilizing the IC9C reporter construct were performed as described before [33, 44]. Surface sterilized seeds were sown on GM medium and incubated for one week. 40 plantlets of each line were transferred into halved Petri dishes containing 10 ml liquid GM medium. After one week of incubation, plants were histochemically GUS stained as described. Then the blue sectors per plant were counted using a binocular microscope to determine the HR rate.
Root growth assays
For the analysis of root growth, seeds were grown vertically under axenic conditions on square plates containing GM medium with 1% plant agar. For root length assays, plants were incubated for 10 days, pictures were taken and image analysis was performed using the ImageJ plugin SmartRoot . For the analysis of cell death in the root meristem, plants were incubated for 5 days followed by propidium iodide staining as previously described .
Quantitative Real Time PCR
For the quantification of 45S and 5S rDNA copy number, quantitative Real Time PCR was performed, as previously described . Therefore, genomic DNA was isolated from 2-week-old plantlets grown in axenic culture. For qRT-PCR analysis, the LightCycler 480 KAPA SYBR Fast Mastermix (Sigma-Aldrich, Steinheim, Germany) was used in total reaction volumes of 10 μl (5 μl KAPA SYBR Fast Mastermix, 0.5 μl of each primer [10 μM], 0.5 ng gDNA, filled up with distilled water). The analysis was performed using a Light Cycler 480 (Roche Diagnostics, Mannheim, Germany). Data was normalized by amplification of the Ubiquitin 10 gene. For primer sequences see S3 Table.
Fluorescence in situ hybridisation
The cytological quantification of 45S rDNA repeats via fluorescence in situ hybridisation (FISH) was performed as previously described . Images were acquired with identical parameters and the area of 45S rDNA probe signals was quantified using ImageJ with background substraction using the “rolling ball” algorithm and a threshold of 16 standard deviations.
S1 Fig. Characterization of T-DNA insertions from Atfancj mutant lines.
(A) The T-DNA in fancja-1 is inserted in intron 24. (B) The T-DNA insertion in fancjb-1 is positioned in intron 10 and accompanied by a 15 bp genomic deletion. Grey box: T-DNA with left borders (LB), red/green box: adjoining genomic sequences.
S2 Fig. Analysis of genomic mutations in fancja-2 and fancja-3 mutant lines.
Induced mutations in fancja-2 (A) and fancja-3 (B) were determined by sequencing of genomic DNA. Mutant line gDNA sequences were aligned with the wild type (WT) reference; sequences differing from the WT are depicted in red. Both mutant lines harbour complex mutations consisting of a combination of deletions, insertions and substitutions.
S3 Fig. cDNA analysis of fancja-2, fancja-3, fancja-4 and fancja-5 mutant lines.
Induced mutations in the fancja mutant lines were verified by sequencing of cDNA. The mutations in fancja-4 and fancja-5 were identical on gDNA and cDNA level. Mutant line cDNA sequences were aligned with the wild type (WT) reference; sequences differing from the WT are depicted in red. Premature stop codons in frame are featured in a red box. The mutations in all mutant lines lead to a frameshift in the open reading frame, resulting in a premature stop codon.
S4 Fig. cDNA analysis of fancjb-2 and fancjb-3 mutant lines.
Induced mutations in the fancjb mutant lines were verified by sequencing of cDNA. The induced mutations were identical on gDNA and cDNA level. Mutant line cDNA sequences were aligned with the wild type (WT) reference; sequences differing from the WT are depicted in red. Premature stop codons in frame are featured in a red box. The mutations in both mutant lines lead to a frameshift in the open reading frame, resulting in a premature stop codon.
S5 Fig. Fertility analysis of fancja-1 and fancjb-1 mutant lines.
Average silique length (A) and seeds per silique (B) were determined for fancja-1 and fancjb-1 mutant lines in comparison to wild type (WT) plants. Fertility of both mutant lines did not differ from the WT. Statistical differences were calculated using the two-tailed t-test with unequal variances: ns = not significant.
The authors want to thank Julia Kremer and Miriam Mielke for excellent technical assistance and Amy Whitbread for critically reading the manuscript.
- 1. Cantor SB, Bell DW, Ganesan S, Kass EM, Drapkin R, Grossman S, et al. BACH1, a novel helicase-like protein, interacts directly with BRCA1 and contributes to its DNA repair function. Cell. 2001; 105: 149–160. pmid:11301010
- 2. Levitus M, Waisfisz Q, Godthelp BC, de Vries Y, Hussain S, Wiegant WW, et al. The DNA helicase BRIP1 is defective in Fanconi anemia complementation group J. Nat Genet. 2005; 37: 934–935. pmid:16116423
- 3. Levran O, Attwooll C, Henry RT, Milton KL, Neveling K, Rio P, et al. The BRCA1-interacting helicase BRIP1 is deficient in Fanconi anemia. Nat Genet. 2005; 37: 931–933. pmid:16116424
- 4. Litman R, Peng M, Jin Z, Zhang F, Zhang J, Powell S, et al. BACH1 is critical for homologous recombination and appears to be the Fanconi anemia gene product FANCJ. Cancer Cell. 2005; 8: 255–265. pmid:16153896
- 5. Kim H, D’Andrea AD. Regulation of DNA cross-link repair by the Fanconi anemia/BRCA pathway. Genes Dev. 2012; 26: 1393–1408. pmid:22751496
- 6. Nepal M, Che R, Zhang J, Ma C, Fei P. Fanconi Anemia Signaling and Cancer. Trends Cancer. 2017; 3: 840–856. pmid:29198440
- 7. Sasaki MS, Tonomura A. A high susceptibility of Fanconi’s anemia to chromosome breakage by DNA cross-linking agents. Cancer Res. 1973; 33: 1829–1836. pmid:4352739
- 8. Gupta R, Sharma S, Sommers JA, Jin Z, Cantor SB, Brosh RM. Analysis of the DNA substrate specificity of the human BACH1 helicase associated with breast cancer. J Biol Chem. 2005; 280: 25450–25460. pmid:15878853
- 9. Cantor S, Drapkin R, Zhang F, Lin Y, Han J, Pamidi S, et al. The BRCA1-associated protein BACH1 is a DNA helicase targeted by clinically relevant inactivating mutations. Proc Natl Acad Sci U S A. 2004; 101: 2357–2362. pmid:14983014
- 10. Dohrn L, Salles D, Siehler SY, Kaufmann J, Wiesmüller L. BRCA1-mediated repression of mutagenic end-joining of DNA double-strand breaks requires complex formation with BACH1. Biochem J. 2012; 441: 919–926. pmid:22032289
- 11. Sommers JA, Rawtani N, Gupta R, Bugreev DV, Mazin AV, Cantor SB, et al. FANCJ uses its motor ATPase to destabilize protein-DNA complexes, unwind triplexes, and inhibit RAD51 strand exchange. J Biol Chem. 2009; 284: 7505–7517. pmid:19150983
- 12. Bharti SK, Awate S, Banerjee T, Brosh RM. Getting Ready for the Dance. FANCJ Irons Out DNA Wrinkles. Genes (Basel). 2016; 7.
- 13. Wu Y, Shin-ya K, Brosh RM. FANCJ helicase defective in Fanconia anemia and breast cancer unwinds G-quadruplex DNA to defend genomic stability. Mol Cell Biol. 2008; 28: 4116–4128. pmid:18426915
- 14. Dhar S, Brosh RM. BLM’s balancing act and the involvement of FANCJ in DNA repair. Cell Cycle. 2018; 17: 2207–2220. pmid:30209988
- 15. Suhasini AN, Rawtani NA, Wu Y, Sommers JA, Sharma S, Mosedale G, et al. Interaction between the helicases genetically linked to Fanconi anemia group J and Bloom’s syndrome. EMBO J. 2011; 30: 692–705. pmid:21240188
- 16. Knoll A, Higgins JD, Seeliger K, Reha SJ, Dangel NJ, Bauknecht M, et al. The Fanconi anemia ortholog FANCM ensures ordered homologous recombination in both somatic and meiotic cells in Arabidopsis. Plant Cell. 2012; 24: 1448–1464. pmid:22547783
- 17. Kurzbauer M-T, Pradillo M, Kerzendorfer C, Sims J, Ladurner R, Oliver C, et al. Arabidopsis thaliana FANCD2 Promotes Meiotic Crossover Formation. Plant Cell. 2018; 30: 415–428. pmid:29352063
- 18. Crismani W, Girard C, Froger N, Pradillo M, Santos JL, Chelysheva L, et al. FANCM limits meiotic crossovers. Science. 2012; 336: 1588–1590. pmid:22723424
- 19. Herrmann NJ, Knoll A, Puchta H. The nuclease FAN1 is involved in DNA crosslink repair in Arabidopsis thaliana independently of the nuclease MUS81. Nucleic Acids Res. 2015; 43: 3653–3666. pmid:25779053
- 20. Dangel NJ, Knoll A, Puchta H. MHF1 plays Fanconi anaemia complementation group M protein (FANCM)-dependent and FANCM-independent roles in DNA repair and homologous recombination in plants. Plant J. 2014; 78: 822–833. pmid:24635147
- 21. Recker J, Knoll A, Puchta H. The Arabidopsis thaliana homolog of the helicase RTEL1 plays multiple roles in preserving genome stability. Plant Cell. 2014; 26: 4889–4902. pmid:25516598
- 22. Vannier J-B, Pavicic-Kaltenbrunner V, Petalcorin MIR, Ding H, Boulton SJ. RTEL1 dismantles T loops and counteracts telomeric G4-DNA to maintain telomere integrity. Cell. 2012; 149: 795–806. pmid:22579284
- 23. Ballew BJ, Yeager M, Jacobs K, Giri N, Boland J, Burdett L, et al. Germline mutations of regulator of telomere elongation helicase 1, RTEL1, in Dyskeratosis congenita. Hum Genet. 2013; 132: 473–480. pmid:23329068
- 24. Deng Z, Glousker G, Molczan A, Fox AJ, Lamm N, Dheekollu J, et al. Inherited mutations in the helicase RTEL1 cause telomere dysfunction and Hoyeraal-Hreidarsson syndrome. Proc Natl Acad Sci U S A. 2013; 110: E3408–16. pmid:23959892
- 25. Le Guen T, Jullien L, Touzot F, Schertzer M, Gaillard L, Perderiset M, et al. Human RTEL1 deficiency causes Hoyeraal-Hreidarsson syndrome with short telomeres and genome instability. Hum Mol Genet. 2013; 22: 3239–3249. pmid:23591994
- 26. Walne AJ, Vulliamy T, Kirwan M, Plagnol V, Dokal I. Constitutional mutations in RTEL1 cause severe dyskeratosis congenita. Am J Hum Genet. 2013; 92: 448–453. pmid:23453664
- 27. Ding H, Schertzer M, Wu X, Gertsenstein M, Selig S, Kammori M, et al. Regulation of murine telomere length by Rtel. An essential gene encoding a helicase-like protein. Cell. 2004; 117: 873–886.
- 28. Barber LJ, Youds JL, Ward JD, McIlwraith MJ, O’Neil NJ, Petalcorin MIR, et al. RTEL1 maintains genomic stability by suppressing homologous recombination. Cell. 2008; 135: 261–271. pmid:18957201
- 29. Hu Z, Cools T, Kalhorzadeh P, Heyman J, de Veylder L. Deficiency of the Arabidopsis helicase RTEL1 triggers a SOG1-dependent replication checkpoint in response to DNA cross-links. Plant Cell. 2015; 27: 149–161. pmid:25595823
- 30. Röhrig S, Schröpfer S, Knoll A, Puchta H. The RTR Complex Partner RMI2 and the DNA Helicase RTEL1 Are Both Independently Involved in Preserving the Stability of 45S rDNA Repeats in Arabidopsis thaliana. PLoS Genet. 2016; 12: e1006394. pmid:27760121
- 31. Mannuss A, Dukowic-Schulze S, Suer S, Hartung F, Pacher M, Puchta H. RAD5A, RECQ4A, and MUS81 have specific functions in homologous recombination and define different pathways of DNA repair in Arabidopsis thaliana. Plant Cell. 2010; 22: 3318–3330. pmid:20971895
- 32. Bagherieh-Najjar MB, de Vries OMH, Hille J, Dijkwel PP. Arabidopsis RecQI4A suppresses homologous recombination and modulates DNA damage responses. Plant J. 2005; 43: 789–798. pmid:16146519
- 33. Hartung F, Suer S, Puchta H. Two closely related RecQ helicases have antagonistic roles in homologous recombination and DNA repair in Arabidopsis thaliana. Proc Natl Acad Sci U S A. 2007; 104: 18836–18841. pmid:18000056
- 34. Klemm T, Mannuß A, Kobbe D, Knoll A, Trapp O, Dorn A, et al. The DNA translocase RAD5A acts independently of the other main DNA repair pathways, and requires both its ATPase and RING domain for activity in Arabidopsis thaliana. Plant J. 2017; 91: 725–740. pmid:28509359
- 35. Chen I-P, Mannuss A, Orel N, Heitzeberg F, Puchta H. A homolog of ScRAD5 is involved in DNA repair and homologous recombination in Arabidopsis. Plant Physiol. 2008; 146: 1786–1796. pmid:18310306
- 36. Kobbe D, Kahles A, Walter M, Klemm T, Mannuss A, Knoll A, et al. AtRAD5A is a DNA translocase harboring a HIRAN domain which confers binding to branched DNA structures and is required for DNA repair in vivo. Plant J. 2016; 88: 521–530.
- 37. Dorn A, Röhrig S, Papp K, Schröpfer S, Hartung F, Knoll A, et al. The topoisomerase 3α zinc-finger domain T1 of Arabidopsis thaliana is required for targeting the enzyme activity to Holliday junction-like DNA repair intermediates. PLoS Genet. 2018; 14: e1007674.
- 38. Hartung F, Suer S, Bergmann T, Puchta H. The role of AtMUS81 in DNA repair and its genetic interaction with the helicase AtRecQ4A. Nucleic Acids Res. 2006; 34: 4438–4448. pmid:16945961
- 39. Enderle J, Dorn A, Beying N, Trapp O, Puchta H. The protease WSS1A, the endonuclease MUS81 and the phosphodiesterase TDP1 are involved in independent pathways of DNA-protein crosslink repair in plants. Plant Cell. 2019:
- 40. Kobbe S, Trapp O, Knoll A, Manuss A, Puchta H. The Translesion Polymerase ζ Has Roles Dependent on and Independent of the Nuclease MUS81 and the Helicase RECQ4A in DNA Damage Repair in Arabidopsis. Plant Physiol. 2015; 169: 2718–2729.
- 41. Klepikova AV, Kasianov AS, Gerasimov ES, Logacheva MD, Penin AA. A high resolution map of the Arabidopsis thaliana developmental transcriptome based on RNA-seq profiling. Plant J. 2016; 88: 1058–1070. pmid:27549386
- 42. Schiml S, Fauser F, Puchta H. The CRISPR/Cas system can be used as nuclease for in planta gene targeting and as paired nickases for directed mutagenesis in Arabidopsis resulting in heritable progeny. Plant J. 2014; 80: 1139–1150. pmid:25327456
- 43. Steinert J, Schiml S, Fauser F, Puchta H. Highly efficient heritable plant genome engineering using Cas9 orthologues from Streptococcus thermophilus and Staphylococcus aureus. Plant J. 2015; 84: 1295–1305. pmid:26576927
- 44. Molinier J, Ries G, Bonhoeffer S, Hohn B. Interchromatid and interhomolog recombination in Arabidopsis thaliana. Plant Cell. 2004; 16: 342–352. pmid:14729918
- 45. Kratz K, Schöpf B, Kaden S, Sendoel A, Eberhard R, Lademann C, et al. Deficiency of FANCD2-associated nuclease KIAA1018/FAN1 sensitizes cells to interstrand crosslinking agents. Cell. 2010; 142: 77–88. pmid:20603016
- 46. Ciccia A, McDonald N, West SC. Structural and Functional Relationships of the XPF/MUS81 Family of Proteins. Annu Rev Biochem. 2008; 77: 259–287. pmid:18518821
- 47. Smogorzewska A, Desetty R, Saito TT, Schlabach M, Lach FP, Sowa ME, et al. A genetic screen identifies FAN1, a Fanconi anemia-associated nuclease necessary for DNA interstrand crosslink repair. Mol Cell. 2010; 39: 36–47. pmid:20603073
- 48. Fontebasso Y, Etheridge TJ, Oliver AW, Murray JM, Carr AM. The conserved Fanconi anemia nuclease Fan1 and the SUMO E3 ligase Pli1 act in two novel Pso2-independent pathways of DNA interstrand crosslink repair in yeast. DNA Repair (Amst). 2013; 12: 1011–1023.
- 49. Schröpfer S, Kobbe D, Hartung F, Knoll A, Puchta H. Defining the roles of the N-terminal region and the helicase activity of RECQ4A in DNA repair and homologous recombination in Arabidopsis. Nucleic Acids Res. 2014; 42: 1684–1697. pmid:24174542
- 50. Blastyák A, Pintér L, Unk I, Prakash L, Prakash S, Haracska L. Yeast Rad5 protein required for postreplication repair has a DNA helicase activity specific for replication fork regression. Mol Cell. 2007; 28: 167–175. pmid:17936713
- 51. Hoege C, Pfander B, Moldovan G-L, Pyrowolakis G, Jentsch S. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature. 2002; 419: 135–141. pmid:12226657
- 52. Ulrich HD, Jentsch S. Two RING finger proteins mediate cooperation between ubiquitin-conjugating enzymes in DNA repair. EMBO J. 2000; 19: 3388–3397. pmid:10880451
- 53. Prakash S, Prakash L. Translesion DNA synthesis in eukaryotes. A one- or two-polymerase affair. Genes Dev. 2002; 16: 1872–1883.
- 54. Nelson JR, Lawrence CW, Hinkle DC. Thymine-thymine dimer bypass by yeast DNA polymerase zeta. Science. 1996; 272: 1646–1649. pmid:8658138
- 55. Sakamoto A, Lan VTT, Hase Y, Shikazono N, Matsunaga T, Tanaka A. Disruption of the AtREV3 gene causes hypersensitivity to ultraviolet B light and gamma-rays in Arabidopsis. Implication of the presence of a translesion synthesis mechanism in plants. Plant Cell. 2003; 15: 2042–2057.
- 56. Wang S, Wen R, Shi X, Lambrecht A, Wang H, Xiao W. RAD5a and REV3 function in two alternative pathways of DNA-damage tolerance in Arabidopsis. DNA Repair (Amst). 2011; 10: 620–628.
- 57. Geuting V, Kobbe D, Hartung F, Dürr J, Focke M, Puchta H. Two distinct MUS81-EME1 complexes from Arabidopsis process Holliday junctions. Plant Physiol. 2009; 150: 1062–1071. pmid:19339504
- 58. Larin M, Gallo D, Tamblyn L, Yang J, Liao H, Sabat N, et al. Fanconi anemia signaling and Mus81 cooperate to safeguard development and crosslink repair. Nucleic Acids Res. 2014; 42: 9807–9820. pmid:25056314
- 59. Rudolf J, Makrantoni V, Ingledew WJ, Stark MJR, White MF. The DNA repair helicases XPD and FancJ have essential iron-sulfur domains. Mol Cell. 2006; 23: 801–808. pmid:16973432
- 60. Wu Y, Suhasini AN, Brosh RM. Welcome the family of FANCJ-like helicases to the block of genome stability maintenance proteins. Cell Mol Life Sci. 2009; 66: 1209–1222. pmid:19099189
- 61. Curtis MJ, Hays JB. Tolerance of dividing cells to replication stress in UVB-irradiated Arabidopsis roots: Requirements for DNA translesion polymerases η and ζ. DNA Repair (Amst). 2007; 6: 1341–1358.
- 62. Fulcher N, Sablowski R. Hypersensitivity to DNA damage in plant stem cell niches. Proc Natl Acad Sci U S A. 2009; 106: 20984–20988. pmid:19933334
- 63. Matsuzaki K, Borel V, Adelman CA, Schindler D, Boulton SJ. FANCJ suppresses microsatellite instability and lymphomagenesis independent of the Fanconi anemia pathway. Genes Dev. 2015; 29: 2532–2546. pmid:26637282
- 64. Barthelemy J, Hanenberg H, Leffak M. FANCJ is essential to maintain microsatellite structure genome-wide during replication stress. Nucleic Acids Res. 2016; 44: 6803–6816. pmid:27179029
- 65. Fransz P, Armstrong S, Alonso-Blanco C, Fischer TC, Torres-Ruiz RA, Jones G. Cytogenetics for the model system Arabidopsis thaliana. Plant J. 1998; 13: 867–876. pmid:9681023
- 66. Murata M, Heslop-Harrison JS, Motoyoshi F. Physical mapping of the 5S ribosomal RNA genes in Arabidopsis thaliana by multi-color fluorescence in situ hybridization with cosmid clones. Plant J. 1997; 12: 31–37. pmid:9263450
- 67. Bridge WL, Vandenberg CJ, Franklin RJ, Hiom K. The BRIP1 helicase functions independently of BRCA1 in the Fanconi anemia pathway for DNA crosslink repair. Nat Genet. 2005; 37: 953–957. pmid:16116421
- 68. Youds JL, Barber LJ, Ward JD, Collis SJ, O’Neil NJ, Boulton SJ, et al. DOG-1 is the Caenorhabditis elegans BRIP1/FANCJ homologue and functions in interstrand cross-link repair. Mol Cell Biol. 2008; 28: 1470–1479. pmid:18086896
- 69. Kumlehn J, Pietralla J, Hensel G, Pacher M, Puchta H. The CRISPR/Cas revolution continues. From efficient gene editing for crop breeding to plant synthetic biology. J Integr Plant Biol. 2018; 60: 1127–1153.
- 70. Yoshikiyo K, Kratz K, Hirota K, Nishihara K, Takata M, Kurumizaka H, et al. KIAA1018/FAN1 nuclease protects cells against genomic instability induced by interstrand cross-linking agents. Proc Natl Acad Sci U S A. 2010; 107: 21553–21557. pmid:21115814
- 71. Panneerselvam J, Wang H, Zhang J, Che R, Yu H, Fei P. BLM promotes the activation of Fanconi Anemia signaling pathway. Oncotarget. 2016; 7: 32351–32361. pmid:27083049
- 72. Daee DL, Ferrari E, Longerich S, Zheng X, Xue X, Branzei D, et al. Rad5-dependent DNA repair functions of the Saccharomyces cerevisiae FANCM protein homolog Mph1. J Biol Chem. 2012; 287: 26563–26575. pmid:22696213
- 73. Peng M, Cong K, Panzarino NJ, Nayak S, Calvo J, Deng B, et al. Opposing Roles of FANCJ and HLTF Protect Forks and Restrain Replication during Stress. Cell Rep. 2018; 24: 3251–3261. pmid:30232006
- 74. Niedzwiedz W, Mosedale G, Johnson M, Ong CY, Pace P, Patel KJ. The Fanconi anaemia gene FANCC promotes homologous recombination and error-prone DNA repair. Mol Cell. 2004; 15: 607–620. pmid:15327776
- 75. Xie J, Litman R, Wang S, Peng M, Guillemette S, Rooney T, et al. Targeting the FANCJ-BRCA1 interaction promotes a switch from recombination to poleta-dependent bypass. Oncogene. 2010; 29: 2499–2508. pmid:20173781
- 76. Bhagwat N, Olsen AL, Wang AT, Hanada K, Stuckert P, Kanaar R, et al. XPF-ERCC1 participates in the Fanconi anemia pathway of cross-link repair. Mol Cell Biol. 2009; 29: 6427–6437. pmid:19805513
- 77. Olivier M, Charbonnel C, Amiard S, White CI, Gallego ME. RAD51 and RTEL1 compensate telomere loss in the absence of telomerase. Nucleic Acids Res. 2018; 46: 2432–2445. pmid:29346668
- 78. Copenhaver GP, Pikaard CS. RFLP and physical mapping with an rDNA-specific endonuclease reveals that nucleolus organizer regions of Arabidopsis thaliana adjoin the telomeres on chromosomes 2 and 4. Plant J. 1996; 9: 259–272. pmid:8820610
- 79. Pruitt RE, Meyerowitz EM. Characterization of the genome of Arabidopsis thaliana. J Mol Biol. 1986; 187: 169–183. pmid:3701864
- 80. Goffová I, Vágnerová R, Peška V, Franek M, Havlová K, Holá M, et al. Roles of RAD51 and RTEL1 in telomere and rDNA stability in Physcomitrella patens. Plant J. 2019.
- 81. Simon L, Rabanal FA, Dubos T, Oliver C, Lauber D, Poulet A, et al. Genetic and epigenetic variation in 5S ribosomal RNA genes reveals genome dynamics in Arabidopsis thaliana. Nucleic Acids Res. 2018; 46: 3019–3033. pmid:29518237
- 82. Mozgová I, Mokros P, Fajkus J. Dysfunction of chromatin assembly factor 1 induces shortening of telomeres and loss of 45S rDNA in Arabidopsis thaliana. Plant Cell. 2010; 22: 2768–2780. pmid:20699390
- 83. Pontvianne F, Blevins T, Chandrasekhara C, Feng W, Stroud H, Jacobsen SE, et al. Histone methyltransferases regulating rRNA gene dose and dosage control in Arabidopsis. Genes Dev. 2012; 26: 945–957. pmid:22549957
- 84. Sarkies P, Murat P, Phillips LG, Patel KJ, Balasubramanian S, Sale JE. FANCJ coordinates two pathways that maintain epigenetic stability at G-quadruplex DNA. Nucleic Acids Res. 2012; 40: 1485–1498. pmid:22021381
- 85. Sims J, Copenhaver GP, Schloegelhofer P. Meiotic DNA repair in the nucleolus employs a non-homologous end joining mechanism. bioRxiv. 2019: 553529.
- 86. Sauer M, Paeschke K. G-quadruplex unwinding helicases and their function in vivo. Biochem Soc Trans. 2017; 45: 1173–1182. pmid:28939694
- 87. Burge S, Parkinson GN, Hazel P, Todd AK, Neidle S. Quadruplex DNA. Sequence, topology and structure. Nucleic Acids Res. 2006; 34: 5402–5415. pmid:17012276
- 88. Porreca RM, Glousker G, Awad A, Matilla Fernandez MI, Gibaud A, Naucke C, et al. Human RTEL1 stabilizes long G-overhangs allowing telomerase-dependent over-extension. Nucleic Acids Res. 2018; 46: 4533–4545. pmid:29522136
- 89. Frizzell A, Nguyen JHG, Petalcorin MIR, Turner KD, Boulton SJ, Freudenreich CH, et al. RTEL1 inhibits trinucleotide repeat expansions and fragility. Cell Rep. 2014; 6: 827–835. pmid:24561255
- 90. Cheung I, Schertzer M, Rose A, Lansdorp PM. Disruption of dog-1 in Caenorhabditis elegans triggers deletions upstream of guanine-rich DNA. Nat Genet. 2002; 31: 405–409. pmid:12101400
- 91. Kruisselbrink E, Guryev V, Brouwer K, Pontier DB, Cuppen E, Tijsterman M. Mutagenic capacity of endogenous G4 DNA underlies genome instability in FANCJ-defective C. elegans. Curr Biol. 2008; 18: 900–905. pmid:18538569
- 92. London TBC, Barber LJ, Mosedale G, Kelly GP, Balasubramanian S, Hickson ID, et al. FANCJ is a structure-specific DNA helicase associated with the maintenance of genomic G/C tracts. J Biol Chem. 2008; 283: 36132–36139. pmid:18978354
- 93. Sun H, Karow JK, Hickson ID, Maizels N. The Bloom’s syndrome helicase unwinds G4 DNA. J Biol Chem. 1998; 273: 27587–27592. pmid:9765292
- 94. van Wietmarschen N, Merzouk S, Halsema N, Spierings DCJ, Guryev V, Lansdorp PM. BLM helicase suppresses recombination at G-quadruplex motifs in transcribed genes. Nat Commun. 2018; 9: 271. pmid:29348659
- 95. Urawa H, Hidaka M, Ishiguro S, Okada K, Horiuchi T. Enhanced homologous recombination caused by the non-transcribed spacer of the rDNA in Arabidopsis. Mol Genet Genomics. 2001; 266: 546–555. pmid:11810225
- 96. Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, et al. Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science. 2003; 301: 653–657. pmid:12893945
- 97. Kleinboelting N, Huep G, Kloetgen A, Viehoever P, Weisshaar B. GABI-Kat SimpleSearch: new features of the Arabidopsis thaliana T-DNA mutant database. Nucleic Acids Res. 2012; 40: D1211–5. pmid:22080561
- 98. Lobet G, Pages L, Draye X. A novel image-analysis toolbox enabling quantitative analysis of root system architecture. Plant Physiol. 2011; 157: 29–39. pmid:21771915