Research Article: A DOG’s View of Fanconi Anemia: Insights from C. elegans

Date Published: May 30, 2012

Publisher: Hindawi Publishing Corporation

Author(s): Martin Jones, Ann Rose.


C. elegans provides an excellent model system for the study of the Fanconi Anemia (FA), one of the hallmarks of which is sensitivity to interstrand crosslinking agents. Central to our understanding of FA has been the investigation of DOG-1, the functional ortholog of the deadbox helicase FANCJ. Here we review the current understanding of the unique role of DOG-1 in maintaining stability of G-rich DNA in C. elegans and explore the question of why DOG-1 animals are crosslink sensitive. We propose a dynamic model in which noncovalently linked G-rich structures form and un-form in the presence of DOG-1. When DOG-1 is absent but crosslinking agents are present the G-rich structures are readily covalently crosslinked, resulting in increased crosslinks formation and thus giving increased crosslink sensitivity. In this interpretation DOG-1 is neither upstream nor downstream in the FA pathway, but works alongside it to limit the availability of crosslink substrates. This model reconciles the crosslink sensitivity observed in the absence of DOG-1 function with its unique role in maintaining G-Rich DNA and will help to formulate experiments to test this hypothesis.

Partial Text

The helicase, FANCJ, is required for the Fanconi Anemia (FA) pathway to function properly and thus maintain genome integrity. In humans, FANCJ mutations have been identified in early-onset breast cancer patients [1, 2] and FA complementation group J patients [3–5]. However, the role of FANCJ in the FA pathway of DNA repair is not fully understood. Some insights have been gained from research on DOG-1 (Deletions Of G-rich DNA), the Caenorhabditis elegans functional ortholog of FANCJ [6–9]. However, even in this relatively simple model system, important questions remain. An outstanding issue is the relationship between the relatively well-known function of DOG-1/FANCJ in preventing replication blocks at unresolved secondary structures and its function in resistance to interstrand crosslinks (ICLs). Previous work from our group has shown that DOG-1 acts upstream of, or parallel to, FCD-2 in the maintenance of G-tracts [7] but is dispensable for FCD-2 focus formation in response to ICL generating agents [8]. One possibility is that DOG-1 takes on two different functions, one in G4 DNA resolution and one in FA crosslink repair. On the other hand, it is possible that its ability to unwind G-rich secondary structure may be sufficient to explain its role in both situations. Here we summarize the current understanding of DOG-1/FANCJ function and hypothesize how to reconcile the two known roles for this protein with its helicase function.

DOG-1 was discovered as being essential for the maintenance of G-rich DNA [6] and was subsequently shown to be the functional ortholog of FANCJ [8]. The value of C. elegans as a model for Fanconi Anemia and ICL repair has been thoroughly reviewed in Youds et al. [9]. An understanding of DOG-1’s role in replication and repair began with the observation that it is a mutator. This was immediately recognizable in C. elegans because of the appearance of spontaneous morphological mutants (described in Cheung et al. [6]) and further explored by the capture and characterization of mutational changes in genes essential for survival (lethal mutations) maintained using a genetic balancer [10]. In dog-1 mutants, the manifestation of the morphological Vab (Variable ABnormal) phenotype was linked to the gene vab-1. An examination of the molecular nature of the vab-1 mutations revealed small deletions that were detectable by PCR. These deletions initiated at the 5′ end of poly-C or the 3′ end of poly-G stretches of DNA and extended for variable distances. These observations led to the proposal that the deletions were occurring as a result of structural blocks to lagging-strand synthesis [6]. In this model, poly-G stretches present in the C. elegans genome form secondary structures. These secondary structures require the helicase function of DOG-1 to resolve them, allowing fork progression. In the absence of the helicase function, deletions are formed between the stalled fork and the upstream Okazaki fragment initiation. Another research group subsequently confirmed the prediction that Okazaki-sized deletions occurred on the lagging strand by using unbiased array comparative hybridization (aCGH) of DOG-1-minus genomes [11].In this study, it was shown that deletions occurred exclusively at sequences that could form quadruplex structures (G4) at a frequency of 4% per site per animal generation. In the human genome, there are estimated to be >300,000 G4 forming sites [12], and these have potentially mutagenic properties implicated in development of cancer susceptibility in the absence of FANCJ function.

Repair pathways that compensate for the absence of DOG-1 in C. elegans have been identified. These include homologous recombination (HR) repair and translesion synthesis (TLS), but not nonhomologous end joining (NHEJ) [7]. In human cell lines, monoubiquitylation of FANCD2 is followed by HR repair. Our genetic analysis has shown that DOG-1 mutants that are also mutant for FCD-2 (FANCD2) exacerbate G-tract deletions [8], as are the HR repair components, BRD-1 (BARD1), RAD-51 (RAD51), and XPF-1 (XPF). Similarly, DOG-1 mutants lacking the TLS polymerases, POL eta and POL kappa have significantly more PCR-detectable G-tract deletions than DOG-1 by itself. That the FA pathway and its downstream repair mechanisms are capable of resolving some G-tract-associated secondary structures in the absence of DOG-1 function indicates that the FA pathway is parallel to DOG-1, at least with respect to the maintenance of G-tracts.

A diagnostic feature of FA defects is the cross-link sensitivity of cultured cells. The presence of ICLs can result in error-prone repair leading to chromosomal instability (CIN) and cell death. In C. elegans, the absence of DOG-1 also results in sensitivity to ICL-inducing agents such as UVA-activated trimethylpsoralen, nitrogen mustard, and cisplatin, but not to X-rays or UVC [8]. Treatment of DOG-1-deficient animals with ICL agents can result in checkpoint-induced cell cycle arrest and apoptosis of germ cells, as well as chromatin bridges and breaks [8]. In response to ICL treatments, animal’s doubly mutant for DOG-1 and FCD-2 are as equally sensitive as each of the single mutants, potentially placing the helicase function of DOG-1 in the same pathway as FCD-2 [8]. Furthermore, DOG-1 is not required for RAD-51 or FCD-2 foci formation after replication stress or ICL induction, possibly placing DOG-1 downstream of FCD-2. This data correlates with that reported by Bridge et al. [17] who demonstrated that FANCJ mutant DT40 cells are also not defective for FANCD2 focus formation.




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