An Arabidopsis FANCJ helicase homologue is required for DNA crosslink repair and rDNA repeat stability

Abstract

Proteins of the Fanconi Anemia (FA) complementation group are required for crosslink (CL) repair in humans and their loss leads to severe pathological phenotypes. Here we characterize a homolog of the Fe-S cluster helicase FANCJ in the model plant Arabidopsis, AtFANCJB, and show that it is involved in interstrand CL repair. It acts at a presumably early step in concert with the nuclease FAN1 but independently of the nuclease AtMUS81, and is epistatic to both error-prone and error-free post-replicative repair in Arabidopsis. The simultaneous knock out of FANCJB and the Fe-S cluster helicase RTEL1 leads to induced cell death in root meristems, indicating an important role of the enzymes in replicative DNA repair. Surprisingly, we found that AtFANCJB is involved in safeguarding rDNA stability in plants. In the absence of AtRTEL1 and AtFANCJB, we detected a synergetic reduction to about one third of the original number of 45S rDNA copies. It is tempting to speculate that the detected rDNA instability might be due to deficiencies in G-quadruplex structure resolution and might thus contribute to pathological phenotypes of certain human genetic diseases.

Fig 1. Gene structure of FANCJ homologs in Arabidopsis thaliana.

AtFANCJA is located at the locus At1g20750 and comprises of 25 exons (red boxes) and 24 introns (black bars) in 5697 bp. The T-DNA insertion in fancja-1 is integrated in intron 24. The mutations in fancja-2/3 are located spanning intron 16/exon 17, the insertions in fancja-4/5 are inserted in exon 17. AtFANCJB (At1g20720) consists of 7137 bp in 30 exons (green boxes) and 29 introns (black bars). In fancjb-1, a T-DNA is inserted in intron 10, fancjb-2 harbours a deletion in exon 16 and fancjb-3 contains a deletion in exon 9.

Fig 2. Sensitivity analysis of fancja and fancjb mutant lines after MMC treatment and HR analysis in fancjb mutant lines.

The fresh weight of the individual lines in response to MMC treatment was determined in relation to untreated control plants. Five independent assays were performed and mean values with standard deviations (error bars) were calculated. (A) The relative fresh weight of all fancja mutant lines did not differ significantly from WT plants. (B) All three fancjb mutant lines depicted a significantly reduced fresh weight compared to WT plants at 5, 10 and 15 μg/ml MMC concentrations. (C) Both fancja fancjb double mutant lines were hypersensitive against MMC treatment, on a level comparable to the fancjb single mutant. (D) Homologous recombination frequency was determined with the IC9C reporter in three independent assays and mean values with standard deviations (error bars) were calculated. The number of blue sectors per plant in both fancjb mutant lines did not differ from WT plants. Statistical differences were calculated using the two-tailed t-test with unequal variances: * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant.

Fig 3. Sensitivity analysis of fancjb-1 fan1-1 and fancjb-1 recq4A-4 double mutants in response to MMC treatment.

Relative fresh weight of double mutants, the respective single mutants and wild-type plants (WT) was determined after MMC treatment. Independent assays were performed at least 4 times and mean values with standard deviations (error bars) were calculated. (A) The relative fresh weight of fancjb-1 fan1-1 double mutants was comparable to both single mutant lines in all tested MMC concentrations. (B) The fancjb-1 recq4A-4 double mutants exhibited a relative fresh weight that did not significantly differ from fancjb-1 single mutants. Statistical differences were calculated using the two-tailed t-test with unequal variances: * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant.

Fig 4. Sensitivity analysis of fancjb-1 rad5A-2, fancjb-1 rev3-5 and fancjb-1 mus81-1 double mutants in response to MMC treatment.

Relative fresh weight of double mutants in comparison to the corresponding single mutants and wild-type plants (WT) was determined after MMC treatment. At least 4 independent assays were performed and mean values with standard deviations (error bars) were calculated. (A) The relative fresh weight of fancjb-1 rad5A-2 double mutants was comparable to the rad5A-2 single mutant in all tested MMC concentrations. (B) The fancjb-1 rev3-5 double mutant exhibited a relative fresh weight that was not significantly different from the rev3-5 single mutant after treatment with MMC. (C) In fancjb-1 mus81-1 double mutants, a significantly reduced fresh weight compared to both single mutants could be observed after 5 and 10 μg/ml MMC treatment. Statistical differences were calculated using the two-tailed t-test with unequal variances: * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant.

Fig 5. Epistasis analysis of FANCJB and RTEL1 in interstrand CL repair, root length and cell death analysis.

(A) Relative fresh weight of fancjb-1 rtel1-1 double mutants, the respective single mutants and wild-type plants (WT) was determined after MMC treatment (n = 3). The fancjb-1 rtel1-1 double mutant depicted a significantly reduced fresh weight compared to both single mutants after treatment with 10 and 15 μg/ml MMC. (B) Root length of 10-day-old fancjb-1 rtel1-1 double mutants was determined in comparison to the respective single mutants and WT plants (n = 4). The roots of fancjb-1 rtel1-1 double mutants were significantly shorter than roots of either single mutant. (C) Cell death analysis in the root meristem of fancjb-1 rtel1-1 double mutants in comparison to the corresponding single mutants and WT plants (n = 5, scale bar = 50 μm). In fancjb-1 rtel1-1 double mutants, a significantly increased amount of dead transiently amplifying (TA) cells, in comparison to both single mutants, could be observed. Statistical differences were calculated using the two-tailed t-test with unequal variances: * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant.

Fig 6. Analysis of rDNA repeat number in fancjb-1 rtel1-1 double mutants.

(A) The rDNA repeat amount was determined by quantitative real-time PCR in the fancjb-1 rtel1-1 double mutant and corresponding single mutants, in relation to WT plants (n = 5). The 45S rDNA repeat number was determined using individual primer pairs for the 18S, 5.8S and 25S rDNA sequences. While the 45S rDNA copy number in the fancjb-1 single mutant was comparable to WT plants, the fancjb-1 rtel1-1 double mutant exhibited a copy number significantly reduced to both single mutants. The 5S rDNA repeat number also showed a significant reduction in the fancjb-1 rtel1-1 double mutant compared to both single mutants. (B) The reduction in 45S rDNA amount was additionally quantified by FISH (fluorescence in situ hybridization), using an Atto488 labelled 45S rDNA probe and DAPI staining of chromatin (n = 4). The area of the 45S rDNA was significantly reduced in the fancjb-1 rtel1-1 double mutant compared to both single mutants. (C) Exemplarily chosen nuclei from each genotype depicting chromatin (blue) with the four 45S rDNA loci (green, scale bar = 10 μm). Statistical differences were calculated using the two-tailed t-test with unequal variances: * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant.

Fig 7. Classification of FANCJB into the interstrand CL repair network of Arabidopsis thaliana.

FANCJB acts together with FAN1 in interstrand CL (ICL) repair, most likely upstream of three subpathways defined by RECQ4A, REV3 and RAD5A. FANCJB and MUS81 act in independent pathways, whereby RTEL1 could be classified into a common pathway with MUS81, separate from FANCJB.