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. 2015 Oct 16;169(4):2718–2729. doi: 10.1104/pp.15.00806

Figure 1.

Figure 1.

Mutations in REV3 motifs led to different effects in DNA damage repair. A, The depicted constructs were assembled from three fragments amplified from Col-0 genomic DNA (gDNA) and cDNA (promoter: 718-bp gDNA, middle fragment: 5,113 bp cDNA, 3′open reading frame (ORF)-terminator gDNA: 1,465 bp) and transformed in rev3-2 mutants. Expression of the REV3 wild-type (WT) construct leads to a protein identical to natural REV3. Point mutations were inserted into motif A and motif C, respectively, with substitution of an Asp residue in the motif to an Ala (marked red in the illustration). These motifs are part of the six conserved regions within the polymerase domain of REV3. The relative fresh weight of homozygous single-locus lines transformed with the wild-type construct of REV3 (B), D1385A (C), and D1549A (D) point mutations in comparison with the wild-type and rev3-2 plants after treatment with MMC, MMS, and cisplatin is shown. For each construct, four different transgenic lines with rev3-2 background are shown. Plantlets were treated with the different genotoxins, and their fresh weight was measured and calculated in relation to the untreated control plants of the same line. Each assay was performed at least three times for calculation of the sd (error bars). B, Hypersensitivity of rev3-2 can be complemented by expression of the wild-type construct, which is true for all tested chemical genotoxins. A control line expressing the construct in a wild-type background (WT::REV3#1) exhibits a relative fresh weight that does not differ from the untransformed wild-type fresh weight. C, Hypersensitivity of rev3-2 cannot be complemented by expression of the D1385A construct, which is true for all tested chemical genotoxins. A control line expressing the construct in a wild-type background (WT::REV3 D1385A#1) exhibits a relative fresh weight that does not differ from the untransformed wild-type fresh weight. Expression of the D1549A construct can complement sensitivity of rev3-2 after treatment with MMS, whereas hypersensitivity is not abolished after treatment with MMC or high concentrations of cisplatin. The line rev3-2::REV3 D1549A#1 behaves like rev3-2 in all tested conditions. A control line expressing the construct in a wild-type background (WT::REV3 D1549A#1) exhibits a relative fresh weight that does not differ from the untransformed wild-type fresh weight. ***, P < 0.001; **, 0.01 > P > 0.001; *, 0.05 > P > 0.01.