
==== Front
Nucleic Acids Res
Nucleic Acids Res
nar
Nucleic Acids Research
0305-1048
1362-4962
Oxford University Press

39011897
10.1093/nar/gkae617
gkae617
AcademicSubjects/SCI00010
Genome Integrity, Repair and Replication
The critical role of the iron–sulfur cluster and CTC components in DOG-1/BRIP1 function in Caenorhabditis elegans
https://orcid.org/0000-0001-5372-2590
Li Xiao Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, Alberta, T2N 4N1, Canada
Department of Medical Genetics, Cumming School of Medicine, University of Calgary, Calgary, Alberta, T2N 4N1, Canada
Alberta Children’s Hospital Research Institute, University of Calgary, Calgary, Alberta, T2N 4N1, Canada

https://orcid.org/0000-0002-9129-570X
Perdomo Ivette Maria Menendez Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, Alberta, T2N 4N1, Canada
Department of Medical Genetics, Cumming School of Medicine, University of Calgary, Calgary, Alberta, T2N 4N1, Canada
Alberta Children’s Hospital Research Institute, University of Calgary, Calgary, Alberta, T2N 4N1, Canada

Rodrigues Alves Barbosa Victoria Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, Alberta, T2N 4N1, Canada
Department of Medical Genetics, Cumming School of Medicine, University of Calgary, Calgary, Alberta, T2N 4N1, Canada
Alberta Children’s Hospital Research Institute, University of Calgary, Calgary, Alberta, T2N 4N1, Canada

Diao Catherine Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, Alberta, T2N 4N1, Canada
Department of Medical Genetics, Cumming School of Medicine, University of Calgary, Calgary, Alberta, T2N 4N1, Canada
Alberta Children’s Hospital Research Institute, University of Calgary, Calgary, Alberta, T2N 4N1, Canada

https://orcid.org/0000-0002-4472-8584
Tarailo-Graovac Maja Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, Alberta, T2N 4N1, Canada
Department of Medical Genetics, Cumming School of Medicine, University of Calgary, Calgary, Alberta, T2N 4N1, Canada
Alberta Children’s Hospital Research Institute, University of Calgary, Calgary, Alberta, T2N 4N1, Canada

To whom correspondence should be addressed. Tel: +1 403 210 9395; Email: maja.tarailograovac@ucalgary.ca
09 9 2024
16 7 2024
16 7 2024
52 16 95869595
02 7 2024
26 6 2024
15 3 2024
© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.
2024
https://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

FANCJ/BRIP1, initially identified as DOG-1 (Deletions Of G-rich DNA) in Caenorhabditis elegans, plays a critical role in genome integrity by facilitating DNA interstrand cross-link repair and resolving G-quadruplex structures. Its function is tightly linked to a conserved [4Fe–4S] cluster-binding motif, mutations of which contribute to Fanconi anemia and various cancers. This study investigates the critical role of the iron–sulfur (Fe–S) cluster in DOG-1 and its relationship with the cytosolic iron–sulfur protein assembly targeting complex (CTC). We found that a DOG-1 mutant, expected to be defective in Fe–S cluster binding, is primarily localized in the cytoplasm, leading to heightened DNA damage sensitivity and G-rich DNA deletions. We further discovered that the deletion of mms-19, a nonessential CTC component, also resulted in DOG-1 sequestered in cytoplasm and increased DNA damage sensitivity. Additionally, we identified that CIAO-1 and CIAO-2B are vital for DOG-1’s stability and repair functions but unlike MMS-19 have essential roles in C. elegans. These findings confirm the CTC and Fe–S cluster as key elements in regulating DOG-1, crucial for genome integrity. Additionally, this study advances our understanding of the CTC’s role in Fe–S protein regulation and development in C. elegans, offering a model to study its impact on multicellular organism development.

Graphical Abstract

Graphical Abstract

Alberta Children’s Hospital Foundation 10.13039/501100003206 Cumming School of Medicine, University of Calgary 10.13039/100012866 Office of Research Infrastructure Programs 10.13039/100016958 P40 OD01440
==== Body
pmcIntroduction

The genome’s integrity is paramount for the proper functioning and preservation of cellular life. The FANCJ/BRIP1 protein plays an instrumental role in protecting genomic fidelity through its involvement in DNA interstrand cross-link (ICL) repair and the resolution of G-quadruplex (G4) structures (1). As a member of the Rad3-like SF2 DNA helicase family, FANCJ/BRIP1 is characterized by a conserved [4Fe–4S] cluster-binding motif comprising four cysteine residues within its helicase domain (2). Pathogenic variants in FANCJ/BRIP1 are associated with Fanconi anemia (FA), a rare hereditary chromosome instability disorder, and are also implicated in hereditary breast and ovarian cancer (3,4). In a seminal study by Odermatt et al. (5), it was elucidated that certain FA and cancer-associated mutations in the Fe–S domain adversely affect Fe–S coordination, compromising ICL repair and G4 structure resolution, thereby underscoring the clinical significance of the Fe–S domain (5). FANCJ/BRIP1 is part of a broader ensemble of cytosolic and nuclear Fe–S proteins, several of which are integral to DNA replication and genome maintenance (6,7). The insertion of the Fe–S cluster into FANCJ/BRIP1 is thought to be mediated by the cytosolic iron–sulfur protein assembly (CIA) targeting complex (CTC), which facilitates interaction with apo-proteins in the cytosol (8–10). Studies in human cells suggest that CTC binding to BRIP1/FANCJ is required in iron–sulfur (Fe–S) cluster acquisition and its protein stability (8,10).

FANCJ/BRIP1 was first identified in Caenorhabditis elegans as DOG-1 (Deletions Of G-rich DNA), where it plays an essential role in preserving G-rich DNA throughout the genome (11–13). The functional parallels between DOG-1 and FANCJ/BRIP1, particularly in the preservation of G-rich DNA and ICL repair, have been substantiated by subsequent research (11,13). Since the identification of DOG-1 in 2002, genetic studies employing a knockout strain have undeniably established C. elegans as an invaluable model for understanding ICL repair, G4 DNA maintenance, and overall molecular mechanisms of FA (11–18).

In this study, we investigate the functional conservation of the Fe–S cluster in DOG-1 by examining a mutant that is expected to be defective in Fe–S cluster binding, as well as mutants in core components of the CTC. We demonstrate a conserved essential role of the Fe–S for proper DOG-1 localization and function. Furthermore, we establish a conserved role of the CTC components, CIAO-1, CIAO-2B and MMS-19, and show that while equally critical for proper DOG-1 localization, stability and function, the CTC components are not equally essential for development in C. elegans.

Overall, this study unveils a previously underappreciated facet of DOG-1 regulation predicated on Fe–S acquisition and cellular localization, orchestrated by the CTC. Thus, we have established a valuable model to study the role of Fe–S clusters in other proteins and the role of CTC in a multicellular organism. This study has implications for understanding the molecular intricacies of how Fe–S clusters and the CTC govern cellular processes that are crucial for genomic integrity and the development of a multicellular organism (19,20).

Materials and methods

Strains

Caenorhabditis elegans strains are obtained from the Caenorhabditis Genetics Center (CGC) and/or generated in the lab (Supplementary Table S3). Strains were maintained on Escherichia coli OP50 seeded NGM plates at 20°C as outlined by Brenner (21), unless reported otherwise. Brood size determination was conducted by placing individual L4 worms on separate plates and transferring them to a new plate every day for 4 days and counting progeny.

Mutant generation

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9)-mediated gene editing was used for mutant generation as detailed by Dokshin et al. (22). For a list of guide RNAs and single-stranded DNA repair templates, please refer to Supplementary Table S4.

TMP-UVA assay

One-day-old adults were treated with 10 μg/ml TMP (trioxsalen, Sigma, T6137-100MG) in M9 buffer for 1 h. After TMP treatment, the worms were moved to a fresh plate and exposed to ultraviolet A (UVA; 200–400 J) at an intensity of 300 μW/cm2 [J/(s m2)]. The treated worms were allowed to recover overnight and then transferred to fresh plates to lay eggs for 4 h (20–24 h after TMP/UVA treatment). The hatch rate was scored 24–48 h after egg laying.

Western blot analysis

Worm protein lysates were prepared by placing 20 one-day-old adult worms in 20 μl of M9 medium, and then denatured by adding 4× sodium dodecyl sulfate sample buffer and boiling for 10 min. Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membranes, and probed with mouse anti-FLAG (1:10 000, Sigma) and mouse anti-α-tubulin (1:10 000, Sigma). Horseradish peroxidase-conjugated donkey anti-rabbit was used as the secondary antibody. Detection was accomplished using ECL Prime Western Blotting Detection Reagents (Cytiva, RPN2232). Bands were visualized using an Amersham Imager 600 and quantified using ImageJ.

Cytological preparation and immunostaining

One-day-old adult worms were collected, washed in M9, fixed in cold methanol, rehydrated with phosphate-buffered saline and mounted in SlowFade Gold Antifade Mountant with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Invitrogen). Dissection and immunostaining of gonads were performed using an adapted protocol as described (23). A mouse anti-FLAG (1:500, Sigma) antibody was used to probe FLAG::DOG-1 or its variant expression in the germ cells from gonads dissected from 1-day-old worms. All images were captured on a Zeiss Imager M2 microscope using Zen software. Nucleocytoplasmic distribution of FLAG::DOG-1 was determined using ImageJ line scan analysis.

Detection of G4 DNA locus deletion

Deletion of endogenous G4 DNA loci was assessed using a polymerase chain reaction (PCR)-based approach as previously described (13,15,24). Protease K-treated worm lysates from single worms were subjected to nested rounds of PCRs with primers that flank a G4 motif. A typical wild-type amplicon is around 1 kb, and smaller PCR bands indicate deletions. The following primers were used to detect a previously characterized G4 locus: qua830 5′-CTAGTTCAGGGTATCTGGAC-3′; 5′-GATTGCGGGCACTTTACCTCG-3′; 5′-CCTTCTCTCGAAGCGCGACC-3′; and 5′-GATTTTATTGACTCTCCGTCCG-3′.

Results

Fe–S cluster is essential for the DOG-1 function

Like human BRIP1/FANCJ, DOG-1 contains a conserved helicase domain and an Fe–S binding domain (Supplementary Figure S1A). To directly understand the significance of Fe–S in DOG-1, we generated a dog-1(C278S) mutant through CRISPR/Cas9-mediated homologous recombination repair. In this mutant, a conserved cysteine (C278) coordinating Fe–S is changed to a serine (Supplementary Figure S1A). This alteration is likely to render DOG-1(C278S) incapable of binding to the Fe–S cluster based on previous studies (5). Concurrently, we created a helicase-deficient dog-1 mutant, dog-1(K121R), to serve as a control (Supplementary Figure S1A). We assessed the proficiency of these mutants in repairing ICLs, employing an established assay in which a deletion mutant dog-1(gk10) has previously exhibited a heightened sensitivity to TMP/UVA (11). We discovered that both dog-1(C278S) and dog-1(K121R) are sensitive to UVA/TMP treatment at a level comparable to the dog-1(gk10) knockout, indicating that Fe–S is likely essential for DOG-1 function in ICL repair (Figure 1A). We also explored the impact of Fe–S on the integrity of guanine-rich DNA regions. We measured the deletion frequency of a previously well-characterized guanine-rich DNA locus qua830, which forms a G4 structure and is frequently deleted in dog-1(gk10) worms (Figures 1B and 4F, and Supplementary Table S1) (15,25). Notably, both dog-1(C278S) and dog-1(K121R) mutants presented with frequent deletions at the qua830 locus, showing the necessity of Fe–S in maintaining guanine-rich DNA sequences (Figure 1B and Supplementary Table S1). To track the cellular localization of DOG-1, we inserted a sequence that encodes a FLAG tag into the N-terminus of the endogenous dog-1 gene (3xflag::dog-1) (Supplementary Figure S1A). First, we confirmed that the insertion of the 3xflag sequence does not change the normal function of DOG-1 as 3xflag::dog-1 worms repair ICLs at wild-type levels (Supplementary Figure S1D). Next, we showed that FLAG::DOG-1 predominantly localizes in the nucleus and associates with DNA, in line with its role as DNA helicase (Supplementary Figure S1C). We then introduced both C278S and K121R mutations in the 3xflag::dog-1 strain through CRISPR/Cas9-mediated homologous recombination repair. We found that FLAG::DOG-1(C278S) is reduced in total worm lysates, while FLAG::DOG-1(K121R) level is not changed (Supplementary Figure S1B). We carried out immunostaining and imaging of the dissected gonads using the same conditions across the genotypes and found that comparable levels of FLAG::DOG-1(C278S) were present in germline cells, but localized in the cytoplasm (Figure 1C). In contrast, both wild-type DOG-1 and helicase-deficient DOG-1(K121R) displayed normal nuclear localization (Figure 1C). This striking observation suggests that the expected Fe–S binding deficiency results in DOG-1 being sequestered in the cytoplasm, preventing its importation into the nucleus. Furthermore, the Fe–S cluster may play a role in maintaining the protein stability of DOG-1. In conclusion, our results underscore a conserved and essential role for the C278 in the functioning of DOG-1, especially in ICL repair and maintaining the integrity of guanine-rich DNA regions.

Figure 1. Fe–S cluster is essential for DOG-1 helicase. (A) Fe–S binding-defective DOG-1(C278S) is sensitive to TMP/UVA-induced ICLs. Error bars represent the standard error of the mean. (B) Guanine-rich DNA at qua830 locus is frequently deleted in dog-1(C278S) worms. (C) FLAG::DOG-1(C278S) is not imported in the nucleus and is sequestered in the cytosol of C. elegans oocytes. Line scan analysis across an oocyte reveals the nucleocytoplasmic distribution of FLAG::DOG-1 (upper) relative to the nucleus (bottom).

Differential requirement of CTC components for viability in C. elegans

Building on the understanding that the Fe–S cluster is vital for DOG-1’s function, we next wanted to explore how DOG-1 might interact with the CTC. We hypothesized that DOG-1, which is expected to be unable to bind Fe–S (as in the DOG-1(C278S) variant), could still associate with the CTC but might not be released as an active protein. To assess the involvement of the CTC in modulating DOG-1 activity, it was crucial to investigate the unexplored function of CTC components in C. elegans. Based on protein sequence homology, we were able to identify Y18D10A.9, F45G2.10 and MMS-19 as orthologues of CIAO1, CIAO2B and MMS19, respectively. For clarity in further discussions, Y18D10A.9 was renamed to ciao-1 and F45G2.10 to ciao-2B (Figure 2A and Supplementary Table S2). We obtained and generated CRISPR knockout alleles for these genes: ciao-1(gk5013), ciao-2B(gk5482) and mms-19(ko), subsequently referred to as ciao-1(ko), ciao-2B(ko) and mms-19(ko) (Figure 2A and Supplementary Table S2).

Figure 2. Differential requirement of CTC genes for viability in C. elegans. (A) Schematic representation of ciao-1, ciao-2B and mms-19 genes and mutations. (B) ciao-1(ko) and ciao-2B(ko) homozygotes are viable but sterile. F1 progeny is produced by P0 heterozygotes. (C) Normal brood size for mms-19(ko) over three generations. (D) Reduced germline population (outlined by dotted lines), lack of mature oocytes (arrows), sperms (triangles) and embryos (diamonds) in ciao-1 and ciao-2B and normal germlines in mms-19(ko) worms.

ciao-1 and ciao-2B are required for viability and fertility, while mms-19 is dispensable

The F1 homozygotes of ciao-1(ko) or ciao-2B(ko) developed into adults, likely due to maternal contributions, but exhibited 100% sterility and failed to produce embryos. As a result, these mutants display maternal effect sterility (Figure 2B). This suggests that ciao-1 and ciao-2B are essential for C. elegans’ development. In contrast, mms-19(ko) homozygotes were viable, exhibiting normal brood size and hatch rate under standard culture conditions (Figure 2C and Supplementary Table S2).

ciao-1 and ciao-2B mutants exhibit impaired germline development

We further analyzed the gonads of ciao-1(ko) and ciao-2B(ko) worms and found them to be smaller than normal. Staining with DAPI revealed a diminished population of germ cells in young adult worms. Moreover, mature sperms, oocytes and embryos were absent in the gonads of these mutants (Figure 2D). In contrast, mms-19(ko) worms showed normal-sized gonads and successful development of sperms, oocytes and embryos (Figure 2D). These findings suggest different requirements of CTC components in fertility and propagation.

MMS-19 is required for ICL repair by regulating DOG-1 stability and nuclear localization

mms-19 is required for ICL repair

As ciao-1(ko) and ciao-2B(ko) homozygotes could not be propagated, our initial investigation on DOG-1 function in these mutant worms focused on the viable mms-19(ko) mutants. Our findings revealed that mms-19(ko) worms display a sensitivity level to TMP/UVA comparable to that of dog-1(gk10) worms, with dog-1(gk10);mms-19(ko) double mutants demonstrating an even greater sensitivity to TMP/UVA treatment (Figure 3A). We attribute this enhanced sensitivity of the double mutant to an additive effect of partial loss of function of DOG-1 and compromised function of other Fe–S proteins that may require MMS-19.

Figure 3. MMS-19 is required for ICL repair by regulating DOG-1 stability and nuclear localization. (A) mms-19 is required for ICL repair. (B) FLAG::DOG-1 is reduced in mms-19(ko) total worm lysate. (C) Diminished nuclear FLAG::DOG-1 in germline cells and embryos of mms-19(ko) worms. Line scan analysis across an oocyte reveals the nucleocytoplasmic distribution of FLAG::DOG-1 (upper) relative to the nucleus (bottom).

MMS-19 is required for proper nuclear localization of DOG-1

In the absence of MMS-19, FLAG::DOG-1 is slightly reduced in the total worm lysates (Figure 3B). Interestingly, FLAG::DOG-1 appeared to be evenly distributed between the cytosol and nucleus in mms-19(ko) oocytes, suggesting either cytosolic retention or impaired nuclear import (Figure 3C). A similar reduction in nuclear localization was observed in mms-19(ko) embryos (Supplementary Figure S3A). This cellular phenotype resembles, to an extent, the distribution of DOG-1(C278S) mutant, supporting the lack of ability of the DOG-1(C278S) to bind Fe–S (Figure 1C). This similarity suggests that MMS-19 might facilitate the release of mature DOG-1 and/or assist in its nuclear import/export. Without MMS-19, DOG-1 seems to be confined to the cytosol, possibly undergoing proteasomal degradation, as suggested by the slightly reduced FLAG::DOG-1 levels and diminished immunostaining in the embryos (Figure 3B and Supplementary Figure S3A). However, this phenotype was not as pronounced as that observed for the DOG-1(C278S) mutation (Figures 1C and 3C, and Supplementary Figure S3A). The partial nuclear localization of DOG-1 may indicate that some mature DOG-1 proteins are made and transported into the nucleus, explaining a lack of G-DNA deletions in the mms-19(ko) worms (Supplementary Figure S3B and Supplementary Table S1).

We further examined another mms-19 deletion mutant, an mms-19(ok3345) generated previously by others and available from the CGC but otherwise uncharacterized. We found that this mutant resulted in an in-frame deletion of amino acids (310–455) in MMS-19 (Supplementary Figure S3C and D). Despite this deletion and a temperature-dependent ‘mortal germline (Mrt)’ phenotype (26) (Supplementary Figure S2A), the substrate-binding domain and the CIAO-2B binding domain remained unaffected (Figure 2A), indicating that it could still interact with DOG-1 and function as part of the CTC. Consistent with this, we found normal sensitivity to TMP/UVA treatment and normal nuclear localization (Figure 3A and B, and Supplementary Figure S3B), supporting the idea that the substrate-binding domain and CIAO-2B binding domain in MMS-19 are sufficient for DOG-1 maturation and release in this mutant.

CIAO-1 and CIAO-2B are also required for DOG-1 stability, ICL repair and G-rich sequence maintenance

ciao-1 and ciao-2B are required for DOG-1 stability

Encouraged by our findings with mms-19, we sought to understand the potential roles of CIAO-1 and CIAO-2B in regulating DOG-1 stability and cellular localization. Through meticulous collection, we obtained a sufficient number of the otherwise infertile ciao-1(ko) and ciao-2B(ko) F1 worms. Analysis of these mutants showed a stark reduction in FLAG::DOG-1 levels in germ cells, with an absence of the DNA-associated foci normally seen in wild-type germ cells (Figure 4A and B) and significant depletion of FLAG::DOG-1 in total worm lysates (Figure 4C). These data imply that when CIAO-1 or CIAO-2B binds to DOG-1, it may protect DOG-1 from breaking down. This is suggested by the still noticeable levels of a form of DOG-1, FLAG::DOG-1(C278S), in germ cells (Figure 1C). These results are consistent with earlier findings that some Fe–S cluster proteins are less stable without CTC genes (10,27).

Figure 4. CIAO-1 and CIAO-2B are also required for DOG-1 stability, ICL repair and G-rich sequence maintenance. (A) Lack of DOG-1 foci in ciao-1 and ciao-2B germline cells. (B) Quantification of DNA-associated foci. Error bars represent the standard error of the mean. (C) Immunoblot of FLAG::DOG-1 ciao-1 and ciao-2B worm lysates. (D) ciao-1(R71A) and ciao-2B(E137A) mutants sensitive to TMP/UVA treatment. (E) G-rich DNA deletion is observed in ciao-1(R71A) and ciao-2B(E137A) mutants. (F) Sanger sequencing confirms the G-tract deletion found in panel (E), resembling characteristic deletions found in dog-1(gk10) worm.

ciao-1 and ciao-2B play a role in ICL repair and guanine-rich DNA maintenance

The sterility of ciao-1(ko) and ciao-2B(ko) homozygotes prevented us from testing their sensitivity to UVA/TMP treatment. We generated missense variants ciao-1(R71A) and ciao-2B(E137A) to circumvent this. Based on the recent crystal structure, R71 and E137 are positioned at the interaction interface of CIAO-1 and CIAO-2B and are predicted to interrupt CIAO-1 and CIAO-2B interaction (Supplementary Figure S4A) (28). The homozygous mutants of ciao-1(R71A) and ciao-2B(E137A) showed no apparent phenotypes and are viable (Supplementary Table S2). However, we noticed that ciao-2B(E137A) had a lower hatching rate at 25°C, indicating that it is sensitive to higher temperature (Supplementary Figure S2B and Supplementary Table S2). The DOG-1 protein levels and cellular location seemed normal in ciao-1(R71A) and ciao-2B(E137A) worms (Supplementary Figure S4B and C). We then tested the sensitivity to TMP/UVA treatment at 23°C, a permissive temperature at which untreated ciao-2B(E137A) worms hatch normally. We found ciao-1(R71A) worms to be sensitive to TMP/UVA treatment at levels comparable to dog-1(gk10), and ciao-2B(E137A) also showed sensitivity, albeit to a lesser extent (Figure 4D). These results were further supported when sensitivity to TMP/UVA treatment was tested in strains carrying the double mutations dog-1(gk10) ciao-1(R71A) or dog-1(gk10);ciao-2B(E137A), as shown in Supplementary Figure S5A.

We examined whether guanine-rich DNA is unstable in ciao-1(R71A) and ciao-2B(E137A) worms. We observed deletions of guanine-rich DNA in both ciao-1(R71A) and ciao-2B(E137A) worms (Figure 4E and Supplementary Table S1). Although the frequency of deletions is rare and not statistically significant when compared to the wild-type strain, we confirmed a unique and highly specific deletion signature by Sanger sequencing (13,24) (Figure 4F). In addition, we did not observe a notable increase in the G4 tract deletion frequency in the strains corresponding to the double mutants dog-1(gk10) ciao-1(R71A) and dog-1(gk10);ciao-2B(E137A) compared to the deletion frequency detected for the dog-1(gk10) knockout strain (Supplementary Figure S5B and Supplementary Table S1). Thus, we infer that these two mutations have minimal effects on G4 tract stability.

Overall, we conclude that, like MMS-19, the proper function of CIAO-1 and CIAO-2B is also required to maintain the appropriate DOG-1 protein level and carry out ICL repair to maintain genome stability.

Discussion

Conserved requirement of Fe–S for DOG-1

Our research demonstrated that Fe–S is indispensable for the DNA helicase DOG-1, particularly concerning its crucial functions in repairing ICLs and in maintaining G-rich DNA sequences. The dog-1 mutant, which is presumably defective in Fe–S binding, mimics the dog-1 deletion mutant and helicase-inactive mutant in experiments involving UVA/TMP and G-tract deletions.

Cellular localization of DOG-1 in response to defective Fe–S binding or insertion by CTC

Our study uncovered a novel finding that DOG-1 is mislocalized when there is a defect in the CTC itself, as in the case of mms-19(ko), or the DOG-1 itself cannot accept Fe–S cluster, as expected in the case of DOG-1(C278S). We hypothesize that the CTC might initially bind DOG-1 tightly prior to inserting Fe–S, leading to confinement of DOG-1 in the cytosol. MMS-19 could play a role in the effective release of mature DOG-1 from the CTC and/or its import/export balance in and out of the nucleus. Without MMS-19, DOG-1 may still acquire Fe–S and enter the nucleus to some extent, but it may not be efficiently released from the CTC and/or imported into the nucleus. Thus, without MMS-19, some DOG-1 must still be able to receive Fe–S and get into the nucleus: this is supported by FLAG::DOG-1 seen in the cell nucleus of mms-19(ko) and no G-rich DNA deletions being detected in mms-19(ko) worms. However, DOG-1 may not be efficiently released from the CTC and/or its import/export in and out of the nucleus may be affected in the absence of MMS-19.

DOG-1 protein stability in response to defective Fe–S binding or insertion by CTC

Previous studies found that human and yeast MMS-19 interact with BRIP1/FANCJ and Rad3, respectively, and BRIP1’s protein levels decrease when MMS-19 is depleted. Our study shows that CIAO-1, CIAO-2B and, to a lesser extent, MMS-19 are necessary for the stability of DOG-1 DNA helicase. Furthermore, we noticed lower total protein levels of the presumably Fe–S binding-defective DOG-1(C278S), which supports the hypothesis that proper Fe–S integration is crucial for DOG-1 stability.

Repair of TMP/UVA-induced ICLs

We observed sensitivities to TMP/UVA treatment for mms-19(ko), ciao-1(R71A) and ciao-2B(E137A) as well as in the predicted Fe–S binding-defective mutant dog-1(C278S). Thus, we attribute the increased sensitivity at least partially to the loss of DOG-1 function due to CTC deficiencies. However, it is essential to recognize that the CTC is known to be involved in integrating Fe–S into a variety of DNA repair proteins within the cytoplasm and nucleus (6). Therefore, mutations in ciao-1, ciao-2B or mms-19 could set off a chain reaction of effects that might either directly or indirectly influence the assessments of DOG-1 functions. For example, the double mutant dog-1(gk10);mms-19(ko) exhibits substantially higher sensitivity to TMP/UVA treatment compared to either dog-1(gk10) or mms-19(ko) single mutants. This could be explained by the additive effect with partial loss of DOG-1 function and the loss of function of another Fe–S DNA repair protein such as rtel-1 (29). However, although the double mutant dog-1(gk10) ciao-1(R71A) also exhibited higher sensitivity to the TMP/UVA treatment than the dog-1(gk10) single mutant, the effect was less robust than that observed for the double mutant dog-1(gk10);mms-19(ko), whereas the dog-1(gk10);ciao-2B(E137A) double mutant sensitivity to TMP/UVA-induced ICLs was comparable to that of the dog-1(gk10) single mutant. These results are consistent with the phenotypic differences observed for the strains containing mms-19(ko) versus ciao-1(R71A) or ciao-2B(E137A) single mutations regarding DOG-1 protein levels and cellular localization.

Maintenance of G-rich DNA

The FANCJ/BRIP1 DNA helicase promotes DNA synthesis through G4 structures, which are stable secondary structures formed by G-rich DNA sequences (30,31). In C. elegans, loss of DOG-1 results in deletions of G4 forming homopolymeric dC/dGs (G4 DNA-induced deletions) (13,24). G4 DNA-induced deletion is highly specific to DOG-1 loss of function, as a large genome-wide mutagenesis genetic screen aiming to identify additional mutations leading to G-tract deletion only identified additional dog-1 alleles and no other genes with similar function (18). In our study, we did not observe any deletion events in the viable mms-19(ko) mutants. This result suggests that residual mature DOG-1 is still present in the nucleus as discussed earlier. Despite observing the loss of DOG-1 protein in ciao-1(ko) and ciao-2B(ko) worms (Figure 4A and C), our efforts using single worm PCR (Supplementary Table S1) and bulk analysis (not shown) failed to detect G-tract deletions. This outcome might stem from restricted DNA replication and proliferation in these worms (Figure 2D), possibly hindering the initial formation of deletions since G-tract deletion generation is reliant on DNA replication and mitosis (25). Alternatively, it could be attributed to the possibility of larger deletions occurring beyond the detection capacity of the nested PCR method applied in these worms. We also failed to detect G-tract deletion events in double ciao-1(R71A);ciao-2B(E137A) mutants (Supplementary Table S1). This could potentially be explained by the fact that ciao-1(R71A) is capable of rescuing the temperature-dependent lethality associated with ciao-2B(E137A) (Supplementary Figure S2B). Consequently, the double mutant seems to behave similarly to the wild type rather than exhibiting an exacerbated phenotype. This observation, combined with the finding that the in-frame deletion of mms-19(ok3345) did not lead to TMP/UV-induced ICL sensitivity (Figure 3A) nor G-tract deletions (Supplementary Table S1) but did result in an Mrt phenotype (Supplementary Figure S2), underscores the intricate and complex interplay between the various components of the CIA machinery and their diverse substrates.

Essentiality of CTC genes

CIAO-1 is an essential gene in yeast, flies and plants (32–34). Our results in worms show that ciao-1(ko) homozygous progeny produced from heterozygous mothers can survive to sterile adults with a reduced number of germ cells and a lack of embryos. RNA interference depletion of both maternal and zygotic transcripts leads to 100% embryonic lethality (35). Taken together, we conclude that ciao-1 is also an essential gene in worms. No CIAO-2B mutants in other model organisms have been reported previously. Our results indicate that similar to ciao-1, ciao-2B is an essential gene required for germline and embryonic development in C. elegans. Though mms-19 knockout mice die at the pre-implantation stage, mms-19 is not an essential gene in yeast and plants (33,36,37) Worm mms-19(ko) is phenotypically normal at standard culture conditions over generations, and thus unlike ciao-1 and ciao-2B, mms-19 is not required for viability. The CTC interacts with numerous cytosolic and nuclear Fe–S client proteins (6). We postulated that a differential requirement of each of the CTC components for maturing of these client proteins underlines the difference in the essentiality of mms-19. Specifically, MMS-19 may play a regulatory role in the release and transport of mature Fe–S proteins.

In conclusion, our study provides a novel understanding of the intricate dynamics among the CTC, Fe–S binding and the DOG-1 DNA helicase, elucidating their roles in DNA repair, protein stability and cellular localization. This multicellular organism model not only unravels the functional nuances of the CTC but also offers a valuable platform to dissect its regulatory mechanisms, significantly contributing to the broader comprehension of DNA repair and cellular homeostasis. In particular, the recently reported implication of CTC genes ciao-1 and mms-19 in the onset of lethal neurodegenerative diseases (19,20) highlights the significance of the work presented herein in assisting the study of human disease mechanisms.

Supplementary Material

gkae617_Supplemental_File

Acknowledgements

We extend our gratitude to Drs Don Moerman and Mark Edgeley for generously providing us with the ciao-1 and ciao-2B knockout alleles. Also, we thank Brittany Ngo and Shivania Reddy for their help in the optimization of G-tract deletion PCR methods. We would also like to express our appreciation to the members of the Tarailo-Graovac lab for their valuable insights and constructive discussions. Finally, we thank Marko Graovac for making a graphical abstract and a gonad representation (Figure 2D) for the manuscript. The funding sources did not have a role in the design of the study, collection and interpretation of data, or writing of the manuscript. Some strains were provided by the CGC, which is funded by the NIH Office of Research Infrastructure Programs.

Author contributions: Xiao Li: conceptualization, formal analysis, methodology, validation and writing—original draft. Maja Tarailo-Graovac: conceptualization, supervision, writing, funding acquisition, review and editing. Victoria Rodrigues Alves Barbosa: phenotyping. Catherine Diao: confirmation of the mms-19 in-frame transcript. Ivette Maria Menendez Perdomo: generation of strains, data collection and analysis.

Data availability

The data underlying this article are available in the article and in its online supplementary material.

Supplementary data

Supplementary Data are available at NAR Online.

Funding

Alberta Children’s Hospital Foundation; Cumming School of Medicine, University of Calgary; NIH Office of Research Infrastructure Programs [P40 OD01440].

Conflict of interest statement. None declared.
==== Refs
References

1. Bharti S.K. , SommersJ.A., GeorgeF., KuperJ., HamonF., Shin-yaK., Teulade-FichouM.P., KiskerC., BroshR.M.Jr Specialization among iron–sulfur cluster helicases to resolve G-quadruplex DNA structures that threaten genomic stability. J. Biol. Chem. 2013; 288 :28217–28229.23935105
2. Rudolf J. , MakrantoniV., IngledewW.J., StarkM.J., WhiteM.F. The DNA repair helicases XPD and FancJ have essential iron–sulfur domains. Mol. Cell. 2006; 23 :801–808.16973432
3. Calvo J.A. , FritchmanB., HernandezD., PerskyN.S., JohannessenC.M., PiccioniF., KelchB.A., CantorS.B. Comprehensive mutational analysis of the BRCA1-associated DNA helicase and tumor-suppressor FANCJ/BACH1/BRIP1. Mol. Cancer Res. 2021; 19 :1015–1025.33619228
4. Suhasini A.N. , BroshR.M.Jr Disease-causing missense mutations in human DNA helicase disorders. Mutat. Res. 2013; 752 :138–152.23276657
5. Odermatt D.C. , LeeW.T.C., WildS., JozwiakowskiS.K., RothenbergE., GariK. Cancer-associated mutations in the iron–sulfur domain of FANCJ affect G-quadruplex metabolism. PLoS Genet. 2020; 16 :e1008740.32542039
6. Braymer J.J. , FreibertS.A., Rakwalska-BangeM., LillR. Mechanistic concepts of iron–sulfur protein biogenesis in biology. Biochim. Biophys. Acta Mol. Cell Res. 2021; 1868 :118863.33007329
7. Wachnowsky C. , FidaiI., CowanJ.A. Iron–sulfur cluster biosynthesis and trafficking—impact on human disease conditions. Metallomics. 2018; 10 :9–29.29019354
8. Gari K. , Leon OrtizA.M., BorelV., FlynnH., SkehelJ.M., BoultonS.J MMS19 links cytoplasmic iron–sulfur cluster assembly to DNA metabolism. Science. 2012; 337 :243–245.22678361
9. Paul V.D. , LillR. Biogenesis of cytosolic and nuclear iron–sulfur proteins and their role in genome stability. Biochim. Biophys. Acta. 2015; 1853 :1528–1539.25583461
10. Stehling O. , VashishtA.A., MascarenhasJ., JonssonZ.O., SharmaT., NetzD.J., PierikA.J., WohlschlegelJ.A., LillR. MMS19 assembles iron–sulfur proteins required for DNA metabolism and genomic integrity. Science. 2012; 337 :195–199.22678362
11. Youds J.L. , BarberL.J., WardJ.D., CollisS.J., O’NeilN.J., BoultonS.J., RoseA.M. DOG-1 is the Caenorhabditis elegans BRIP1/FANCJ homologue and functions in interstrand cross-link repair. Mol. Cell. Biol. 2008; 28 :1470–1479.18086896
12. Wu Y. , Shin-yaK., BroshR.M.Jr FANCJ helicase defective in Fanconi anemia and breast cancer unwinds G-quadruplex DNA to defend genomic stability. Mol. Cell. Biol. 2008; 28 :4116–4128.18426915
13. Cheung I. , SchertzerM., RoseA., LansdorpP.M. Disruption of dog-1 in Caenorhabditis elegans triggers deletions upstream of guanine-rich DNA. Nat. Genet. 2002; 31 :405–409.12101400
14. Wilson D.M. , RieckherM., WilliamsA.B., SchumacherB. Systematic analysis of DNA crosslink repair pathways during development and aging in Caenorhabditis elegans. Nucleic Acids Res. 2017; 45 :9467–9480.28934497
15. Koole W. , van SchendelR., KarambelasA.E., van HeterenJ.T., OkiharaK.L., TijstermanM. A polymerase theta-dependent repair pathway suppresses extensive genomic instability at endogenous G4 DNA sites. Nat. Commun. 2014; 5 :3216.24496117
16. Jones M. , RoseA. A DOG’s view of Fanconi anemia: insights from C. elegans. Anemia. 2012; 2012 :323721.22690333
17. Maizels N. Genomic stability: FANCJ-dependent G4 DNA repair. Curr. Biol. 2008; 18 :R613–R614.18644339
18. Kruisselbrink E. , GuryevV., BrouwerK., PontierD.B., CuppenE., TijstermanM. Mutagenic capacity of endogenous G4 DNA underlies genome instability in FANCJ-defective C. elegans. Curr. Biol. 2008; 18 :900–905.18538569
19. Maio N. , OrbachR., ZaharievaI., TopfA., DonkervoortS., MunotP., MuellerJ., WillisT., VermaS., PericS.et al . Loss of function of the cytoplasmic Fe–S assembly protein CIAO1 causes a neuromuscular disorder with compromise of nucleocytoplasmic Fe–S enzymes. 2023; medRxiv doi: 20 December 2023, preprint: not peer reviewed 10.1101/2023.12.20.23300170.
20. van Karnebeek C.D.M. , Tarailo-GraovacM., LeenR., MeinsmaR., CorreardS., Jansen-MeijerJ., PrykhozhijS.V., PenaI.A., BanK., SchockS.et al . CIAO1 and MMS19 deficiency: a lethal neurodegenerative phenotype caused by cytosolic Fe–S cluster protein assembly disorders. Genet. Med. 2024; 26 :101104.38411040
21. Brenner S. The genetics of Caenorhabditis elegans. Genetics. 1974; 77 :71–94.4366476
22. Dokshin G.A. , GhantaK.S., PiscopoK.M., MelloC.C. Robust genome editing with short single-stranded and long, partially single-stranded DNA donors in Caenorhabditis elegans. Genetics. 2018; 210 :781–787.30213854
23. Phillips C.M. , McDonaldK.L., DernburgA.F. Cytological analysis of meiosis in Caenorhabditis elegans. Methods Mol. Biol. 2009; 558 :171–195.19685325
24. Youds J.L. , O’NeilN.J., RoseA.M. Homologous recombination is required for genome stability in the absence of DOG-1 in Caenorhabditis elegans. Genetics. 2006; 173 :697–708.16547095
25. Lemmens B. , van SchendelR., TijstermanM. Mutagenic consequences of a single G-quadruplex demonstrate mitotic inheritance of DNA replication fork barriers. Nat. Commun. 2015; 6 :8909.26563448
26. Ahmed S. , HodgkinJ. MRT-2 checkpoint protein is required for germline immortality and telomere replication in C. elegans. Nature. 2000; 403 :159–164.10646593
27. van Wietmarschen N. , MoradianA., MorinG.B., LansdorpP.M., UringaE.J. The mammalian proteins MMS19, MIP18, and ANT2 are involved in cytoplasmic iron–sulfur cluster protein assembly. J. Biol. Chem. 2012; 287 :43351–43358.23150669
28. Kassube S.A. , ThomaN.H. Structural insights into Fe–S protein biogenesis by the CIA targeting complex. Nat. Struct. Mol. Biol. 2020; 27 :735–742.32632277
29. Barber L.J. , YoudsJ.L., WardJ.D., McIlwraithM.J., O’NeilN.J., PetalcorinM.I., MartinJ.S., CollisS.J., CantorS.B., AuclairM.et al . RTEL1 maintains genomic stability by suppressing homologous recombination. Cell. 2008; 135 :261–271.18957201
30. Bosch P.C. , Segura-BayonaS., KooleW., van HeterenJ.T., DewarJ.M., TijstermanM., KnipscheerP FANCJ promotes DNA synthesis through G-quadruplex structures. EMBO J. 2014; 33 :2521–2533.25193968
31. Wu C.G. , SpiesM. G-quadruplex recognition and remodeling by the FANCJ helicase. Nucleic Acids Res. 2016; 44 :8742–8753.27342280
32. Balk J. , Aguilar NetzD.J., TepperK., PierikA.J., LillR. The essential WD40 protein Cia1 is involved in a late step of cytosolic and nuclear iron–sulfur protein assembly. Mol. Cell. Biol. 2005; 25 :10833–10841.16314508
33. Luo D. , BernardD.G., BalkJ., HaiH., CuiX. The DUF59 family gene AE7 acts in the cytosolic iron–sulfur cluster assembly pathway to maintain nuclear genome integrity in Arabidopsis. Plant Cell. 2012; 24 :4135–4148.23104832
34. Jung J. , YeomE., ChoiK.W. Ciao1 interacts with crumbs and Xpd to regulate organ growth in Drosophila. Cell Death Dis. 2020; 11 :365.32404863
35. Maeda I. , KoharaY., YamamotoM., SugimotoA. Large-scale analysis of gene function in Caenorhabditis elegans by high-throughput RNAi. Curr. Biol. 2001; 11 :171–176.11231151
36. Lombaerts M. , TijstermanM., VerhageR.A., BrouwerJ. Saccharomyces cerevisiae mms19 mutants are deficient in transcription-coupled and global nucleotide excision repair. Nucleic Acids Res. 1997; 25 :3974–3979.9321645
37. Wang Y. , SinghR., TongE., TangM., ZhengL., FangH., LiR., GuoL., SongJ., SrinivasanR.et al . Positional cloning and characterization of the papaya diminutive mutant reveal a truncating mutation in the CpMMS19 gene. New Phytol. 2020; 225 :2006–2021.31733154
