The critical role of the iron–sulfur cluster and CTC components in DOG-1/BRIP1 function in Caenorhabditis elegans

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.


Introduction
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)(9)(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 ).
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, CIA O-1, CIA O-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 ).

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 Esc heric hia 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 / cm 2 [J / (s m 2 )].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 oneday-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 antiα-tubulin (1:10 000, Sigma).Horseradish peroxidaseconjugated 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.

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 S1 A).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 S1 A).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 S1 A).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 1 A).We also explored the impact of Fe-S on the integrity of guanine-rich DNA regions.We measured the deletion frequency of a previously wellcharacterized guanine-rich DNA locus qua830 , which forms a G4 structure and is frequently deleted in dog-1(gk10) worms (Figures 1 B and 4 F, 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 1 B 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 S1 A).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 S1 D).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 S1 C).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 S1 B).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 1 C).In contrast, both wild-type DOG-1 and helicase-deficient DOG-1(K121R) displayed normal nuclear localization (Figure 1 C).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.

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 CIA O1, CIA O2B and MMS19, respectively.For clarity in further discussions, Y18D10A.9 was renamed to ciao-1 and F45G2.10 to ciao-2B (Figure 2 A 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(k o) , ciao-2B(k o) and mms-19(ko) (Figure 2 A and Supplementary Table S2 ).

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 2 B).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 2 C 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 2 D).In contrast, mms-19(ko) worms showed normal-sized gonads and successful development of sperms, oocytes and embryos (Figure 2 D).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 3 A).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.

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 3 B).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 3 C).A similar reduction in nuclear localization was observed in mms-19(ko) embryos ( Supplementary Figure S3 A).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 1 C).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 3 B and Supplementary Figure S3 A).However, this phenotype was not as pronounced as that ob-  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 S3 C and  D).Despite this deletion and a temperature-dependent 'mortal germline (Mrt)' phenotype ( 26 ) ( Supplementary Figure S2 A), the substrate-binding domain and the CIAO-2B binding domain remained unaffected (Figure 2 A), 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 S3 B), 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 regulat-ing 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 4 A and B) and significant depletion of FLAG::DOG-1 in total worm lysates (Figure 4 C).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 1 C).These results are consistent with earlier findings that some Fe-S cluster proteins are less stable without CTC genes ( 10 ,27 ).

ciao-1 and ciao-2B play a role in ICL repair and guaninerich 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 S4 A) ( 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 S2 B 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 S4 B 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 4 D).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 S5 A.
We examined whether guanine-rich DNA is unstable in ciao-1(R71A) and ciao-2B(E137A) worms.We observed dele-tions of guanine-rich DNA in both ciao-1(R71A) and ciao-2B(E137A) worms (Figure 4 E 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 4 F).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 S5 B 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.

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 cy-tosol.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 CIA O-1, CIA O-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 DNAinduced 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 4 A 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 2 D), 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 S2 B).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 inframe deletion of mms-19(ok3345 ) did not lead to TMP / UV-induced ICL sensitivity (Figure 3 A) 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)(33)(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.representation (Figure 2 D) 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.

Figure 1 .
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::D O G-1 (upper) relative to the nucleus (bottom).

Figure 3 .
Figure 3. MMS-19 is required for ICL repair by regulating D O G-1 st abilit y 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).