Elsevier

DNA Repair

Volume 10, Issue 11, 10 November 2011, Pages 1174-1182
DNA Repair

CHL-1 provides an essential function affecting cell proliferation and chromosome stability in Caenorhabditis elegans

https://doi.org/10.1016/j.dnarep.2011.09.011Get rights and content

Abstract

A family of helicases that are important in maintaining genome stability is the iron–sulfur group. Members of this family include DOG-1/FANCJ, RTEL1, XPD and Chl1p/DDX11. In Caenorhabitis elegans, the predicted gene M03C11.2 has orthology to the CHL1 (Chromosome loss 1) gene in Saccharomyces cerevisiae and DDX11 (DEAD/H box polypeptide 11) in humans. In this paper, we show that the chl-1 gene in C. elegans is required for normal development and fertility. Mutants have lineage-independent cell proliferation defects that result in a Stu (sterile uncoordinated) phenotype, characterized by gonadal abnormalities and a reduced number of D motor neurons and seam cells. A chromosome stability defect is present in the germ cells, where an abnormal number of DAPI-staining chromosomes appear in diakinesis. CHL-1 function is required for the integrity of poly-guanine/poly-cytosine DNA in the absence of DOG-1/FANCJ: the loss of CHL-1 alone does not result in the deletion of G-tracts, but it does increase the number of deletions observed in the dog-1; chl-1 double mutant, indicating a role for CHL-1 during replication and repair. In addition, we observed that cohesin defects increased the number of deletions in the absence of DOG-1/FANCJ. Our results demonstrate a role for CHL-1 in cell proliferation and maintaining normal chromosome numbers, and implicate CHL-1 in chromosome stability and repair of unresolved secondary structures during replication.

Highlights

► This study establishes C. elegans as an animal model for the study of CHL-1 function. ► The work demonstrates a role for CHL-1 in repair of unresolved secondary structures. ► CHL-1 has an essential function linking together replication, repair and cohesion.

Introduction

The stability of the genome is essential for normal development and survival in all organisms. Among the many factors that contribute to genome stability are several related helicases defined by the presence of an iron–sulfur domain [1]. There are four core members in this family: the XPD/RAD3 and CHL1/DDX11 helicases in metazoans and yeast, and the FANCJ/DOG-1 and RTEL1 helicases in metazoans [1].

The XPD helicase (xeroderma pigmentosum D) functions in nucleotide excision repair and is a subunit in the TFIIH complex. Patients with mutations in the encoding gene present with extreme sensitivity to ultraviolet exposure known as xeroderma pigmentosum, stemming from defects in the nucleotide excision repair pathway [2]. Another well-studied member of this helicase family is the human FANCJ helicase (Fanconi anemia J) and its ortholog in Caenorhabditis elegans, DOG-1 (deletion of guanine-rich DNA). Defects in these helicases lead to heightened sensitivity to DNA interstrand crosslinks and the spontaneous deletion of guanine-rich DNA [3], [4], [5], [6], [7], [8], [9], [10], [11], [12]. Based on the directionality of these deletions, FANCJ/DOG-1 has been proposed to unwind secondary guanine structures during the replication of the lagging strand [4]. The mouse Rtel1 helicase (regulator of telomere length) and its orthologs in humans (RTEL1) and C. elegans (RTEL-1) represent a third member of this helicase family. They control homologous recombination by disassembling recombination intermediates, and defects in their function result in shortened telomeres and increased meiotic recombination frequency [13], [14], [15], [16].

While much is known about the functions of these three helicases in the family, less is known about the biological function of CHL-1. The CHL1 gene was first identified in yeast in a screen for mutants with decreased fidelity of chromosome transmission [17], [18] and later shown to be required for sister chromatid cohesion [19], [20], [21], [22] and the repair of methylmethane sulfonate-induced DNA lesions [23], [24]. Impaired function in the mammalian ortholog ChlR1 (CHL1-related 1)/DDX11 (DEAD/H box polypeptide 11) caused defects in sister chromatid cohesion in mouse embryos [25] and human cell cultures [26]. Recently, it was shown that biallelic mutations in the human gene resulted in Warsaw breakage syndrome (WABS), a newly described genetic condition characterized by congenital abnormalities, abnormal skin pigmentation and severe growth retardation [27]. At the chromosomal level, this syndrome exhibited sister chromatid cohesion defects similar to the cohesinopathy observed in Roberts syndrome. In addition, DDX11−/− cells were sensitive to mitomycin C (MMC), a DNA interstrand crosslinking agent and camptothecin (CPT), a topoisomerase inhibitor. This sensitivity to replicative stress is similar to that observed in cell lines derived from Fanconi anemia patients [27].

In biochemical assays, the human ChlR1/DDX11 ATPase-helicase co-immunoprecipitated with cohesin subunits [26]. Furthermore, it has been found to possess directional specificity in its helicase function [28]. Its function is also required for the efficient activity of the flap endonuclease Fen1, thus implicating ChlR1/DDX11 in the processing of the lagging strand during replication [28].

Beyond the characterizations of chl1/ChlR1/DDX11 mutant phenotypes and the biochemical characterization of the human ChlR1/DDX11 helicase, little is known about the function of this gene in a multi-cellular organism. C. elegans provides an excellent system for investigating the biological role of CHL-1. The genetic amenability of C. elegans allows us to explore its interaction with DOG-1, with which it shares homology and lagging-strand specificity. Here, we characterize the mutant phenotype of chl-1 and describe a functional interaction with dog-1 in maintaining genome stability.

Section snippets

Bioinformatic analyses of iron–sulfur helicases

The amino acid sequences of the following helicases were obtained from the National Center for Biotechnology Information: from C. elegans, CHL-1 (accession number NP_499295.1), DOG-1 (accession number NP_493618.1) and RTEL-1 (accession number NP_492769.1); from humans, DDX11 (accession number NP_004390.3), FANCJ/BRIP1 (accession number NP_114432.2), RTEL1 (accession number CAC16223.1) and XPD/ERCC2 (accession number AAL48323.1); from Mus musculus, Ddx11 (accession number NP_001003919.1), Fancj

M03C11.2 is the C. elegans ortholog of S. cerevisiae CHL1 and human DDX11

We identified M03C11.2 as a gene whose conceptual translation had a high degree of similarity to the S. cerevisiae Chl1 and the Homo sapiens DDX11 amino acid sequences (Fig. 1A). Chl1 and DDX11 are DEAH-box helicases belonging to a subset of superfamily 2 (SF2) helicases defined by an iron–sulfur domain [1]. The predicted cysteine residues in the iron–sulfur domain and the conserved SF2 helicase motifs are shown in Fig. 1A. The conceptual translation of M03C11.2 is more similar to human DDX11,

Discussion

In this work, we have characterized the Caenorhabditis elegans chl-1 gene and its loss-of-function phenotype. Without CHL-1 function, animals have proliferation defects in the soma and the germline. Chromosome instability can be observed in chl-1 animals that have diakinetic oocytes. We have also shown that CHL-1 function is involved in preventing guanine tract deletions in the absence of DOG-1 function, which establishes C. elegans CHL-1 as an important player in the response to DNA secondary

Conclusions

The chl-1 gene in C. elegans is structurally and functionally conserved with its ortholog in yeast and mammals. The gene product provides a function required for cell proliferation and chromosome stability. In its absence there are reduced cell numbers in both the soma and the germ line. Although not required for G-tract stability, CHL-1 functions to prevent the enhancement of the G-tract deletions observed in the absence of DOG-1/FANCJ, as do components of the cohesin core complex, and the

Conflict of interest

None.

Acknowledgements

Some nematode strains used in this work were provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources (NCRR). We thank the National Bioresource Project (Shohei Mitani) for providing the tm2188 allele of chl-1. Funding was provided by a Canadian Institutes of Health grant to AMR and graduate fellowship support from Natural Sciences and Engineering Council and the University of British Columbia to GC. The authors thank David Baillie, Martin

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