The nuclease FAN1 is involved in DNA crosslink repair in Arabidopsis thaliana independently of the nuclease MUS81.
Bottom Line: No FAN1 homolog is present in Drosophila and Saccharomyces cerevisiae.Both the virus-type replication-repair nuclease and the ubiquitin-binding ubiquitin-binding zinc finger domains are essential for this function.Mutations in both FAN1 and the endonuclease MUS81 resulted in greater sensitivity against CLs than in the respective single mutants.
Affiliation: Botanical Institute II, Karlsruhe Institute of Technology, Hertzstrasse 16, Karlsruhe, 76187, Germany.Show MeSH
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Mentions: We were able to identify in Arabidopsis FAN1 a VRR nuclease domain and a putative UBZ domain. To analyze if these domains are essential for the CL repair function of FAN1 in Arabidopsis, we created different mutation or deletion constructs and analyzed whether they were able to complement the increased sensitivity against MMC observed in fan1-1. To analyze the VRR nuclease domain, we cloned two different constructs each carrying a point mutation to inhibit the nuclease activity of FAN1. These two point mutations have shown in humans to limit the endonuclease activity of FAN1 on branched DNA structures to different extents (5). To amplify these constructs, genomic DNA was used. Both constructs were under the control of the natural FAN1 promoter and terminator. In the first construct, named FAN1 NUC1, the asparagine acid residue at position 833 was replaced by an alanine residue. In the second construct, named FAN1 NUC2, the lysine residue at position 854 was replaced by an alanine residue. In biochemical experiments with the human protein corresponding to the K854A mutant, some minor residual activity was detected. No activity was observed with a protein corresponding to a D833A mutant (see Supplementary Figure S3 in (5)). Both constructs were transformed into the fan1-1 mutant line. Four independent fan1-1::FAN1 NUC1 and fan1-1::FAN1 NUC2 complementation lines were established before sensitivity against MMC was tested. The increased sensitivity of fan1-1 could not be complemented by the FAN1 NUC1 construct; all tested complementation lines had the same fresh weight after MMC treatment as the fan1-1 mutant (Figure 4A). Complementation lines carrying the FAN1 NUC2 construct exhibited an intermediate phenotype, as the hypersensitivity of fan1-1 was complemented only partially (Figure 4B). These findings indicate that the K854A mutation retained some residual nuclease activity, as observed in mammals.
Affiliation: Botanical Institute II, Karlsruhe Institute of Technology, Hertzstrasse 16, Karlsruhe, 76187, Germany.