Arabidopsis UEV1D promotes Lysine-63-linked polyubiquitination and is involved in DNA damage response

Abstract

DNA damage tolerance (DDT) in budding yeast requires Lys-63-linked polyubiquitination of the proliferating cell nuclear antigen. The ubiquitin-conjugating enzyme Ubc13 and the Ubc enzyme variant (Uev) methyl methanesulfonate2 (Mms2) are required for this process. Mms2 homologs have been found in all eukaryotic genomes examined; however, their roles in multicellular eukaryotes have not been elucidated. We report the isolation and characterization of four UEV1 genes from Arabidopsis thaliana. All four Uev1 proteins can form a stable complex with At Ubc13 or with Ubc13 from yeast or human and can promote Ubc13-mediated Lys-63 polyubiquitination. All four Uev1 proteins can replace yeast MMS2 DDT functions in vivo. Although these genes are ubiquitously expressed in most tissues, UEV1D appears to express at a much higher level in germinating seeds and in pollen. We obtained and characterized two uev1d null mutant T-DNA insertion lines. Compared with wild-type plants, seeds from uev1d null plants germinated poorly when treated with a DNA-damaging agent. Those that germinated grew slower, and the majority ceased growth within 2 weeks. Pollen from uev1d plants also displayed a moderate but significant decrease in germination in the presence of DNA damage. This report links Ubc13-Uev with functions in DNA damage response in Arabidopsis.

Figure 1.

Sequence Analysis of At UEV1 Genes and Their Products. (A) Genomic organization of UEV1. Open boxes, untranslated region; closed boxes, coding regions; solid lines, introns; dotted lines, identical intron–exon alignment between different UEV1 genes. (B) Amino acid sequence alignment of At Uev1 and Uevs from six other organisms. The sequences were aligned and edited using the BioEdit program version 5.0.9 (Hall, 1999). Residues are highlighted when 50% or more are identical. Critical residues for Mms2/Uev functions are indicated with asterisks underneath the residues.

Figure 2.

Biochemical Properties of Uev1. (A) Physical interaction between Ubc13 and Uev1 in a yeast two-hybrid assay. The PJ69-4A transformants carrying one Gal4_AD (from pGAD424) and one Gal4_BD (from pGBT9) were replicated onto various plates as indicated and incubated for 3 d or as specified before being photographed. The result is representative of at least five independent transformants from each treatment. (B) Physical interactions between At Uev1A/D and Ubc13 from yeast or human in a yeast two-hybrid assay. Experimental conditions were the same as in (A). (C) Protein interactions between Uev1A/D and Ubc13 by an affinity pull-down assay. Purified GST (lane 5), GST-Uev1A (lane 6), or GST-Uev1D (lane 7) was added to GST microspin columns. Following incubation, the columns were spun and washed, and purified Ubc13A was added to the column. After reincubation and washing, the column contents were eluted with reduced glutathione, followed by SDS-PAGE gel analysis. Lanes 1 to 4 contain purified input proteins as indicated at top. Note that spontaneous cleavage occurred in the two GST-Uev1 protein samples (lanes 3 and 4). (D) Ub conjugation by Ubc13, Uev1A, and Uev1D. An in vitro Ub conjugation assay was performed using purified proteins as indicated. Assay samples were subjected to SDS-PAGE, and a protein gel blot using an anti-Ub antibody was assayed to monitor poly-Ub formation. The low background of spontaneously formed di-Ub in the absence of E2 or Uev (lanes 1, 2, and 6) is commonly observed in these reactions (McKenna et al., 2001).

Figure 3.

Complementation of Yeast mms2 Mutants by At UEV1. (A) Complementation of the mms2 single mutant by At UEV1. WXY942 (mms2Δ) transformants were grown overnight and printed onto YPD and YPD + 0.025% MMS gradient plates. The plates were incubated at 30°C for 2 d before being photographed. The arrow indicates higher MMS concentrations. Several transformants of each treatment were tested with the same result, and only one is shown here. (B) Complementation of the mms2 ubc13 double mutant (WXY955) by At UEV1A, At UEV1D, and At UBC13. Experimental conditions were as in (A).

Figure 4.

Tissue Distribution of UEV1 Expression. (A) Relative expression of UEV1 transcripts in different tissues was determined using data from the Arabidopsis NASCArrays microarray database (http://affymetrix.Arabidopsis.info/narrays/experimentbrowse.pl) (Craigon et al., 2004). R, roots of 17-d plants; S, shoots of 8-d seedlings; L, rosette leaf 2 of 17-d plants; RL, mature rosette leaves of 23-d plants; St, second internode of 21-d plants; F, stage 12 flowers of 21-d plants; P, mature pollen; G3h, seed germinating for 3 h. The original microarray data are from AtGenExpress: Expression Atlas of Arabidopsis Development (TAIR accession number 1006710873: ATGE_9, ATGE_12, ATGE_24, ATGE_27, ATGE_33, ATGE_73, and ATGE_96 samples) (Schmid et al., 2005), except for G3h data, which are from AtGenExpress: Expression Profiling of Early Germinating Seeds (TAIR accession number 1007966994: RIKEN-PRESTON2 sample). (B) Expression of UEV1 transcripts in different tissues analyzed by RT-PCR. The At4g33380 gene was assayed as an input control (Czechowski et al., 2005). The exposure time of the gels is shown at left (BioDoc-It System; UVP). C, cell suspension; R, roots of 13-d seedlings; S, shoots of 13-d seedlings; L3, leaves of 3-week plants; L5, leaves of 5-week plants; St, stems of 5-week plants; F, floral tissues of 5-week plants; G6h and G2d, seeds germinating on Petri dishes for 6 h and 2 d, respectively; P, pollen.

Figure 5.

Confirmation of Two uev1d T-DNA Insertion Mutants. (A) Genomic structure showing the positions of two T-DNA insertions in UEV1D. Open boxes, exons; closed boxes, UEV1D ORF; lines, introns. SP1, 5′ gene-specific primer AtUEV1D-1; SP2, 3′ gene-specific primer AtUEV1D-2; LB1, T-DNA left border primer. (B) and (C) Genomic DNA PCR to confirm uev1d-1 (1d-1) (B) and uev1d-2 (1d-2) (C). The fragment was amplified using three primers (SP1, SP2, and LB1) in each reaction and genomic DNA from Columbia (WT), 1d-1 (B), or 1d-2 (C) as a template. (D) RT-PCR detection of the UEV1 transcripts. UEV1 gene-specific primers were used for RT-PCR against total RNA extracted from Columbia (WT), uev1d-1, and uev1d-2 lines. Total RNA was extracted from flowers.

Figure 6.

Phenotypic Analysis of DNA Damage Response during Seed Germination. (A) to (D) The homozygous uev1d-1 mutant (open triangles) is compared with three controls: Columbia (open squares), an unrelated T-DNA insertion line, SALK_042051 (closed diamonds), and a wild-type segregant line from the same SALK_064912 seeds (1d-1WT; closed circles). Synchronized seeds were sown on half-strength Murashige and Skoog agar plates with or without MMS as indicated and incubated for the given period, and phenotypes were quantitatively assessed. (A) Percentage of seed germination after 5 d. (B) Representative photographs after 13 d. (C) Percentage of seedlings with green cotyledons after 13 d. (D) Relative fresh weight of seedlings with green cotyledons after 13 d. (E) and (F) The homozygous uev1d-2 mutant (open triangles) is compared with 1d-2WT, a wild-type segregant from the same T-DNA insertion line SALK_052144 (closed circles). (E) Percentage of seed germination after 5 d. (F) Percentage of seedlings with green cotyledons after 13 d. All data are averages of three independent experiments with sd.

Figure 7.

Phenotypic Analysis of DNA Damage Response during in Vitro Pollen Germination. (A) Representative in vitro pollen germination images of 1d-1WT and uev1d-1 with or without MMS treatment as indicated. (B) Summary of the pollen germination results. Data presented are averages of three independent experiments with sd. Open bars, 1d-1WT; closed bars, uev1d-1.