Figure 1.
Characterization of T-DNA mutants and 3-AB treated seedlings.
(A) Schematic of PARP proteins. The triangles indicate the position of the T-DNA insertions. The T-DNA for PARP1 is located in the intron between exons 6 and 7, within the PARP catalytic domain. The T-DNA for PARP2 is located in exon 10, within the WGR domain. SAP: SAF-A/B, Acinus and PIAS (nucleic acid-binding domain); WGR: Named after conserved central motif (Trp, Gly, Arg) (putative DNA-binding domain); PARP: PARP regulatory and catalytic domain; PADR1: Domain of unknown function found in PARPs; BRCT: BRCA1 C-terminus. (B) Semi-quantitative RT-PCR for PARP1 and PARP2 expression levels in parp1 (top), parp2 (middle) and parp1 parp2 double mutants (bottom). Actin-2 served as a loading control. (C) Wild type (top) and G4 tert mutant seedlings (bottom) grown in 5 mM 3-AB/0.6% DMSO (left) or in 0.6% DMSO (right). (D) qRT-PCR for XRCC2 expression in 3-AB-treated wild type seedlings relative to untreated seedlings. * = p-value <0.05 by Student’s two-tailed t-test.
Figure 2.
Arabidopsis PARPs respond to genotoxic stress.
(A) Morphological and developmental defects of seedlings grown in increasing concentrations of MMS. Left panel: parp1 and parp2 single mutants. Right panel: parp1 parp2 double mutants. ku70 mutants were used as a positive control for MMS sensitivity. (B) qRT-PCR for PARP expression in wild type seedlings. Seedlings were either treated with 20 µM zeocin/0.6% DMSO or 0.6% DMSO (untreated) for 4 h. PARP3 expression was not detected. * = p-value <0.005 compared to untreated.
Figure 3.
PARP1 and PARP2 negatively regulate expression of each other and PARP3.
qRT-PCR results for PARP1 (A), PARP2 (B), PARP3 (C), and BRCA1 (D) transcripts in seedlings treated with 3-AB and zeocin. All values were calculated relative to wild type seedlings that were not treated with either 3-AB or zeocin. DMSO = no 3-AB; nt = not treated with zeocin; z = zeocin treatment. Student’s two-tailed t-tests were used for statistical analysis. P-values <0.05 are indicated by symbols above the bars for the following comparisons: ‡ = mutant compared to wild type with the same treatment; * = untreated compared to zeocin treated for the same genotype and 3-AB status; § = DMSO compared to 3-AB treated for the same genotype and zeocin status.
Figure 4.
Arabidopsis PARPs respond to the absence of telomerase.
(A) RT-PCR of PARP1 transcript levels in different generations of tert mutants. (B) RT-PCR of PARP2 expression in wild type and 3rd generation (G3) tert mutants at increasing concentrations of MMS.
Figure 5.
Arabidopsis telomerase is not stimulated by PARPs.
(A) TRAP analysis on seedlings. Seedlings were treated with either 3-AB (WT) or DMSO (WT and G4 tert mutants) (B) Quantitative TRAP results for 7-day-old 3-AB-treated wild type seedlings relative to untreated seedlings. P-value = 0.03 by Student’s two-tailed t-test.
Figure 6.
PARPs are not required to prevent end-to-end chromosome fusions.
Telomere fusion PCR for (A) 3-AB treated wild type seedlings, and (B) 3-AB treated tert mutants. The pairs of subtelomeric primers used for TF- PCR are indicated below each blot, where 1L refers to the left arm of chromosome 1 and 2R to the right arm of chromosome 2, etc. ctc1 mutants were used as a positive control.
Figure 7.
PARPs are not required to maintain telomere length in Arabidopsis.
(A) TRF analysis of bulk telomeres in several individual wild type, parp1, parp2, and parp1 parp2 plants. (B–D) PETRA analysis of telomeres on specific chromosome arms in wild type and parp mutants. Primer naming convention is the same as in the Figure 6 legend. (B) Analysis of the telomeres on chromosome arms 1L and 2R. DNA was from pooled seedlings. (C) Analysis of four different chromosome arms for individual parp1, parp2, and parp1 parp2 mutant plants. Chromosome arms tested are listed below the lanes. (D) Analysis of wild type and G4 tert pooled seedlings grown in either 0.6% DMSO or 5 mM 3-AB/0.6% DMSO using the 2R primer. Molecular weight markers are shown to the left of each gel.