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Nucleic Acids Res
2015 Apr 20;437:3653-66. doi: 10.1093/nar/gkv208.
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The nuclease FAN1 is involved in DNA crosslink repair in Arabidopsis thaliana independently of the nuclease MUS81.
Herrmann NJ
,
Knoll A
,
Puchta H
.
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Fanconi anemia is a severe genetic disorder. Mutations in one of several genes lead to defects in DNA crosslink (CL) repair in human cells. An essential step in CL repair is the activation of the pathway by the monoubiquitination of the heterodimer FANCD2/FANCI, which recruits the nuclease FAN1 to the CL site. Surprisingly, FAN1 function is not conserved between different eukaryotes. No FAN1 homolog is present in Drosophila and Saccharomyces cerevisiae. The FAN1 homolog in Schizosaccharomyces pombe is involved in CL repair; a homolog is present in Xenopus but is not involved in CL repair. Here we show that a FAN1 homolog is present in plants and it is involved in CL repair in Arabidopsis thaliana. Both the virus-type replication-repair nuclease and the ubiquitin-binding ubiquitin-binding zinc finger domains are essential for this function. FAN1 likely acts upstream of two sub-pathways of CL repair. These pathways are defined by the Bloom syndrome homolog RECQ4A and the ATPase RAD5A, which is involved in error-free post-replicative repair. Mutations in both FAN1 and the endonuclease MUS81 resulted in greater sensitivity against CLs than in the respective single mutants. These results indicate that the two nucleases define two independent pathways of CL repair in plants.
Figure 1. The genomic structure and the protein domains of AtFAN1. (A) AtFAN1 is composed of 15 exons and 14 introns with a total length of 4382 bp from the start to stop codons. Exons are shown as blue boxes, and introns are shown as gray lines. UTR regions are shown as gray boxes. (B) Domains present in the FAN1 proteins of humans and Arabidopsis. Both contain a VRR nuclease domain (Nuc), a potential UBZ domain and a DNA-binding domain in between [a HIRAN (H) domain in Arabidopsis and a SAP domain in humans]. (C) Alignment of HsFAN1 and AtFAN1 protein sequences reveals several conserved amino acids in the UBZ domain region. Identical amino acids are marked by (‘*’), highly similar aa are marked by (‘:’) and aa that are only weakly similar are marked by (‘.’).
Figure 2. Phylogenetic relationship of FAN1 homologs. Following the alignment of protein sequences by ClustalOmega, a tree was calculated using the maximum likelihood method. The values near the branches represent the percentage of replicate trees in which the associated taxa clustered together in the Bootstrap test (1000 replicates). Pt (Pan troglodytes), Hs (Homo sapiens), Pa (Pongo abelii), Mm (Mus musculus), Md (Monodelphis domestica), Oa (Ornithorhynchus anatinus), Gg (Gallus gallus), Ac (Anolis carolinensis), Dr (Danio rerio), Pp (Physcomitrella patens), Os (Oryza sativa), Atr (Amborella trichopoda), Sl (Solanum lycopersicum), At (Arabidopsis thaliana), Zt (Zymoseptoria tritici), An (Aspergillus nidulans), Sp (Schizosaccharomyces pombe), Um (Ustilago maydis), Pg (Puccinia graminis).
Figure 3. Sensitivity of Atfan1–1 and the fan1-1::FAN1 WT complementation lines after MMC treatment. To calculate relative fresh weights of the tested lines, the absolute fresh weights of MMC-treated plants were normalized with fresh weights of untreated control plants from identical lines. Each assay was performed at least three times to calculate mean values and standard deviations (error bars). (A) Compared to WT plants, the Atfan1-1 mutant showed a reduced relative fresh weight after MMC treatment. (B) The fan1-1::FAN1 WT complementation lines #1, #2, #3 and #4 were able to complement the increased sensitivity of fan1-1 after MMC treatment. P-value ≤ 0.05 (*); P-value < 0.01 (**); P-value < 0.001 (***).
Figure 4. Contribution of FAN1 domains to the repair of MMC-induced DNA lesions. Sensitivity of the fan1-1::FAN1 NUC1, fan1-1::FAN1 NUC2 and the fan1-1::FAN1 Del UBZ complementation lines after MMC treatment. To calculate relative fresh weights of the tested lines, absolute fresh weights of MMC-treated plants were normalized with fresh weights of untreated control plants from identical lines. Each assay was performed at least three times to calculate mean values and standard deviations (error bars). (A) The complementation lines fan1-1::FAN1 NUC1 #1, #2, #3 and #4 showed a relative fresh weight comparable to that of the fan1-1 mutant and were not able to complement the hypersensitivity of fan1-1 against MMC. (B) The complementation lines fan1-1::FAN1 NUC2 #1, #2, #3 and #4 showed an intermediate relative fresh weight compared to WT plants and the fan1-1 mutant. (C) The complementation lines fan1-1::FAN1 Del UBZ #1, #2, #3 and #4 showed a relative fresh weight comparable to the fan1-1 mutant and were not able to complement the increased sensitivity of fan1-1 against MMC. P-value ≤ 0.05 (*); P-value < 0.01 (**); P-value < 0.001 (***).
Figure 5. Analysis of dead cells in the root tips of fan1-1 and WT plants. After staining with PI, the amount of whole root tips exhibiting at least one dead SC was determined. The SCs were differentiated into vascular SC, endodermal SC, epidermal SC, columellaSC, TA cells and cells of the QC. (A) Representative images of confocal planes from PI-stained root tips of fan1-1 and WT plants and the quantification of whole root tips with dead cells under normal conditions. (B) Representative images of confocal planes from PI-stained root tips of fan1-1 and WT plants and the quantification of whole root tips with dead cells after treatment with 2.5 μg/ml MMC.
Figure 6. Sensitivity of different double mutants containing Atfan1-1 after MMC treatment. To calculate the relative fresh weights of the tested lines, the absolute fresh weights of MMC-treated plants were normalized with fresh weights of untreated control plants from identical lines. Each assay was performed at least three times to calculate the mean values and standard deviations (error bars). (A) The relative fresh weight of fan1-1 mus81-1 was decreased compared to the relative fresh weigh of both single mutants. (B) The fan1-1 rad5A-2 double mutant exhibited a relative fresh weight comparable to the fresh weight of the rad5A-2 single mutant. (C) The fan1-1 recq4A-4 double mutant showed a relative fresh weight comparable to that of the fan1-1 single mutant. (D) The relative fresh weight of the fan1-1 mhf1-1 double mutant was similar to that of the fan1-1 single mutant. P-value ≤ 0.05 (*); P-value < 0.01 (**); P-value < 0.001 (***). ns = not significant.
Figure 7. Model of different interstrand CL repair pathways with fan1-1 in A. thaliana. During interstrand CL repair, FAN1 acts above the two sub-pathways defined by RECQ4A and RAD5A. We assume that MHF1 and RAD5A act in the same pathway. Furthermore, MUS81 defines a FAN1-independent ‘backup’ pathway of CL repair in plants.
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