DOG-1 is the Caenorhabditis elegans BRIP1/FANCJ homologue and functions in interstrand cross-link repair

Abstract

Fanconi anemia (FA) is a cancer susceptibility syndrome characterized by defective DNA interstrand cross-link (ICL) repair. Here, we show that DOG-1 is the Caenorhabditis elegans homologue of FANCJ, a helicase mutated in FA-J patients. DOG-1 performs a conserved role in ICL repair, as dog-1 mutants are hypersensitive to ICL-inducing agents, but not to UVC irradiation or X rays. Genetic analysis indicated that dog-1 is epistatic with fcd-2 (C. elegans FANCD2) but is nonepistatic with brc-1 (C. elegans BRCA1), thus establishing the existence of two distinct pathways of ICL repair in worms. Furthermore, DOG-1 is dispensable for FCD-2 and RAD-51 focus formation, suggesting that DOG-1 operates downstream of FCD-2 and RAD-51 in ICL repair. DOG-1 was previously implicated in poly(G)/poly(C) (G/C) tract maintenance during DNA replication. G/C tracts remain stable in the absence of ATL-1, CLK-2 (FA pathway activators), FCD-2, BRC-2, and MLH-1 (associated FA components), implying that DOG-1 is the sole FA component required for G/C tract maintenance in a wild-type background. However, FCD-2 is required to promote deletion-free repair at G/C tracts in dog-1 mutants, consistent with a role for FA factors at the replication fork. The functional conservation between DOG-1 and FANCJ suggests a possible role for FANCJ in G/C tract maintenance in human cells.

FIG. 1.

(A) Protein sequence alignment of C. elegans DOG-1 (CeDOG-1) and human FANCJ (HsFANCJ). Identical amino acids are shown in white on a black background; conserved amino acids are shown in black on a gray background. The sequences were aligned using ClustalW 1.83 and shaded with BoxShade 3.21. (B) Schematic of conserved domains of C. elegans DOG-1, chicken FANCJ (GgFANCJ), and human FANCJ as determined by the Conserved Domains Database. The BRCA1-interacting region of human FANCJ, including Ser990 and Phe993 (shown by the black square), are not conserved in chicken FANCJ or C. elegans DOG-1.

FIG. 2.

dog-1 mutants are not sensitive to DNA damage from UVC or X-ray irradiation but are hypersensitive to ICL-inducing agents. The sensitivities of N2(Wt) and dog-1 animals to UVC irradiation (A), X rays (B), UV-activated TMP (TMP-UVA) (C), and nitrogen mustard (HN2) (D) are shown. All values reported are ± standard error of the mean.

FIG. 3.

Chromosomal aberrations are present, and the DNA damage checkpoint is active, in dog-1 mutants after cross-linking treatment. (A) Representative chromatin bridges (arrows) and breaks (arrowheads) observed in the germ lines of dog-1 mutants following treatment with TMP (10 μg/ml) and UVA (200 J). Quantification of chromosome breakages and bridges in N2(Wt), fcd-2(ok1145), and dog-1(gk10) animals following treatment with TMP-UVA showed similar numbers of breaks and bridges in fcd-2 and dog-1 mutants. (B) Schematic of the C. elegans germ line. Nuclei in the mitotic zone serve as the stem cell compartment of the germ line and undergo cell cycle arrest following DNA damage. Nuclei move through the transition zone, where they take on a characteristic crescent shape as they begin the early stages of meiosis I. Meiosis progresses through pachytene, diplotene, and diakinesis, and completes once the oocyte has been fertilized. Cells in late pachytene/early diplotene undergo apoptosis in response to physiological cues or DNA damage. Early embryos are visible prior to ovulation. (C) Germ line mitotic zone nuclei of N2(Wt), dog-1, and clk-2 mutants stained with DAPI. Enlarged arrested nuclei were observed in the germ line mitotic zones of N2(Wt) and dog-1, but not in clk-2 mutants, following treatment with 40 mM HU or 200 μM nitrogen mustard (HN2). (D) Numbers of SYTO12-stained corpses in N2(Wt) and dog-1 animals with no treatment and 24 h after DNA cross-linking with TMP-UVA treatment (n, number of animals scored for apoptosis).

FIG. 4.

dog-1 is epistatic with fcd-2, but not with brc-1, for sensitivity to ICL-inducing agents. (A) TMP-UVA sensitivities of N2(Wt), dog-1, and fcd-2 mutants and dog-1; fcd-2 double mutants. (B) Nitrogen mustard (HN2) sensitivities of N2(Wt), dog-1, and fcd-2 mutants and dog-1; fcd-2 double mutants. (C) TMP-UVA sensitivities of N2(Wt), dog-1, and brc-1 mutants and dog-1; brc-1 double mutants. All values reported are ± standard error of the mean.

FIG. 5.

FCD-2 and RAD-51 focus formation after DNA damage is unaffected by the absence of DOG-1. (A) Representative images of mitotic zone anti-FCD-2 staining (red) on N2(Wt), dog-1, and fcd-2 mutants following no treatment or treatment with 180 μM cisplatin (CDDP), 200 μM nitrogen mustard (HN2), or 40 mM HU. (B) Representative images of mitotic zone anti-RAD-51 staining (red) on N2(Wt), dog-1, and rfs-1 mutants following no treatment or treatment with 180 μM cisplatin or 200 μM nitrogen mustard. (C) Quantification of mitotic nuclei with RAD-51 foci in N2(Wt), dog-1, and rfs-1 mutants before and after treatment with 180 μM cisplatin.