Telomerase dependence of telomere lengthening in Ku80 mutant Arabidopsis

Abstract

We have identified a ku80 mutant of Arabidopsis and show that telomerase is needed to generate the longer telomeres observed in this mutant. Telomeres are specialized nucleoprotein structures at the ends of chromosomes that permit cells to distinguish chromosome ends from double-strand breaks, thus preventing chromosome fusion events. Ku80 deficiency results in the lengthening of telomeres, a phenotype also seen in an Arabidopsis ku70 mutant. Furthermore, homogeneous populations of ku80 mutant cells show a steady increase in the length of telomere tracts, which reach an equilibrium length and then stabilize. In contrast to that in mammals, Ku80 deficiency in Arabidopsis cells does not cause end-to-end fusion of chromosomes. This telomere lengthening is dependent on the presence of telomerase, although it is not attributable to a significant increase in telomerase activity per se. These results demonstrate the essential role of the Ku80 protein as a negative regulator of telomerase function in plant cells.

Figure 1.

T-DNA Insertion into the Arabidopsis KU80 Gene. Boxes represent the exons in the KU80 gene. The position of the T-DNA insertion is indicated. At bottom, the junction sequences of the inserted T-DNAs are shown. The three boxes show the three junctions: KU80–T-DNA, T-DNA–T-DNA, and T-DNA–KU80. The mutant ku80 genomic DNA sequence is aligned with the KU80 genomic sequence and the ends of the inserted T-DNA. The mutant sequence and the sequences homologous with it (from which it derives) are shown in uppercase letters. Orientations of the T-DNA sequence are indicated as right border–left border (rblb) and left border–right border (lbrb). LB, left border; RB, right border.

Figure 2.

Telomere Dynamics in Homogeneous Populations of ku80 Mutant Dividing Cells. DNA was prepared from wild-type (lanes +) and two independent ku80 (lanes a and b) cell cultures grown in liquid for the indicated number of weeks. MboI-digested DNA was examined by DNA gel blot analysis using the telomeric repeat probe (A). The DNA gel blot was washed off and reprobed with a subtelomeric region from the long arm of chromosome 2 (B), which specifically detects the telomeric MboI fragment of this chromosome arm.

Figure 3.

Arabidopsis ku80 Mutant Telomeres Are Sensitive to Bal31 Exonuclease. Genomic DNA from wild-type (WT) and ku80 mutant cells lines was treated with the indicated amounts (units) of Bal31 endonuclease, digested with MboI, and analyzed by DNA gel blot hybridization with the telomere repeat probe (A). The DNA gel blot was washed off and reprobed with a subtelomeric region from the long arm of chromosome 2 (B), which specifically detects the telomeric MboI fragment of this chromosome arm. ku80 and wild-type DNA were equally sensitive to Bal31 treatment.

Figure 4.

Long Telomeres in ku80 Require Telomerase. DNA gel blot analysis of telomere length using the telomeric repeat probe (A) and the chromosome II subtelomeric probe (B). MboI-digested DNA from wild-type (K+T+), attert mutant (K+t−), ku80 mutant (k−T+), and the ku80 attert double mutant (k−t−) plants was analyzed. Data from two subsequent generations of plants (G1 and G2) are shown.

Figure 5.

Telomerase Activity in Ku−/− Mutant Cells. Protein extracts were prepared from wild-type (+) and homozygous (k) ku80 mutant cells. The fifth lane (−) contained no protein extract. Telomerase activity was measured by the modified TRAP assay as described in Methods. Extracts were pretreated (+) or not (−) with RNase. Band intensity was quantified by phosphorimager analysis and is shown graphically at right for the wild type (+) and the ku80 mutant (ku80).