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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 - Nucleic Acids Res. (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: Botanical Institute II, Karlsruhe Institute of Technology, Hertzstrasse 16, Karlsruhe, 76187, Germany.

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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 (***).
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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 (***).

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.


The nuclease FAN1 is involved in DNA crosslink repair in Arabidopsis thaliana independently of the nuclease MUS81.

Herrmann NJ, Knoll A, Puchta H - Nucleic Acids Res. (2015)

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 (***).
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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 (***).
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.

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.

View Article: PubMed Central - PubMed

Affiliation: Botanical Institute II, Karlsruhe Institute of Technology, Hertzstrasse 16, Karlsruhe, 76187, Germany.

Show MeSH
Related in: MedlinePlus