RAD50 function is essential for telomere maintenance in Arabidopsis

Abstract

We have identified and characterized an Arabidopsis thaliana rad50 mutant plant containing a T-DNA insertion in the AtRAD50 gene and showing both meiotic and DNA repair defects. We report here that rad50/rad50 mutant cells show a progressive shortening of telomeric DNA relative to heterozygous rad50/RAD50 controls and that the mutant cell population rapidly enters a crisis, with the majority of the cells dying. Surviving rad50 mutant cells have longer telomeres than wild-type cells, indicating the existence in plants of an alternative RAD50-independent mechanism for telomere maintenance. These results report the role of a protein essential for double-strand break repair in telomere maintenance in higher eukaryotes.

Figure 1

Comparison of telomeres lengths in DNA prepared from flowers of wild-type and rad50 mutant plants. Southern analysis of flower DNA from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) rad50 mutant plants digested with either MboI (lanes 1–3) or HinfI (lanes 4–6). Positions of molecular weight size markers (in kb) are indicated to the right.

Figure 2

Telomere shortening in homozygous rad50/rad50 mutant cells. DNA was prepared from calli generated from leaves heterozygous (+/−) or homozygous (−/−) for the mutant rad50 allele. DNA was prepared from cells grown in liquid culture for 8 (lanes 1 and 2) or 10 (lanes 3 and 4) weeks. MboI-digested DNA was analyzed by Southern analysis by using the telomeric repeat probe. Positions of molecular weight size markers (in kb) are indicated at the side of each panel (note that the two panels are from two different gels).

Figure 3

Mutant rad50/rad50 cells present a senescent phenotype. rad50 homozygous (Upper) and rad50 heterozygous (Lower) cells of the same age grown on nutrient agar. The rad50 homozygous cells are dying, whereas the heterozygous cells are alive. Sectors of surviving homozygous cells are shown with arrows.

Figure 4

Surviving rad50/rad50 mutant cells present longer telomeres. Southern analysis of MboI-digested DNA using the telomeric repeat probe. DNA was prepared from rad50 heterozygous (+/−) and homozygous (−/−) mutant cells as well as wild-type (+/+) cells after different periods in culture. Positions of molecular weight size markers (in kb) are indicated to the right.

Figure 5

Dynamics of the chromosome II subtelomeric region of rad50/rad50 mutant cells with culture time. MboI-digested DNA was prepared from rad50/rad50 mutant cells grown for 8, 10, 21, 23, and 32 weeks after initiation of cell culture. The Southern blot was probed with the subtelomeric region present in chromosome II. Position of marker sizes are shown to the right of each panel (note that lanes 1, 3, 4, and 5 are from the same gel and that lane 2 is from a reprobing of the filter shown in Fig. 2, lane 4).

Figure 6

Telomerase activity in rad50 mutant cells. Protein extracts were prepared from heterozygous (+/−) and homozygous (−/−) rad50 mutant cells. Telomerase activity was detected by the TRAP assay as described in Materials and Methods. Products were separated on a 10% sequencing gel to reveal the periodic band profile. The TRAP assay was realized with extracts prepared from heterozygous (lanes, 3, 6, and 9) and homozygous (lanes 2, 5, and 8) rad50 mutant cells as indicated at the top of the figure. Lanes 1, 4, and 7 are the no-extract controls. Lanes 1–3 are from the TRAP assay under standard conditions. For, lanes 4–6, cell extracts were treated with RNaseA before the telomerase step. For lanes 7–9, samples were treated with RNaseA after the telomerase step and before the PCR step.