Increased telomere length and hypersensitivity to DNA damaging agents in an Arabidopsis KU70 mutant

Abstract

We have identified a putative homologue of the KU70 gene (AtKU70) from Arabidopsis thaliana. In order to study its function in plants we have isolated an A.thaliana line that contains a T-DNA inserted into AtKU70. Plants homozygous for this insertion appear normal and are fertile. In other organisms the KU70 gene has been shown to play a role in the repair of DNA damage induced by ionising radiation (IR) and by radiomimetic chemicals such as methylmethane sulfonate (MMS). We show that AtKU70(-/-) plants are hypersensitive to IR and MMS, and thus the AtKU70 gene plays a similar role in DNA repair in plants as in other organisms. The KU70 gene also plays a role in maintaining telomere length. Yeast and mammalian cells deficient for Ku70 have shortened telomeres. When we studied the telomeres in the AtKU70(-/-) plants we found unexpectedly that they were significantly longer (>30 kb) than was found in wild-type plants (2-4 kb). We propose several hypotheses to explain this telomere lengthening in the AtKU70(-/-) plants.

Figure 1

The sequences of the A.thaliana (AtKU70), Homo sapiens (HsKU70) and S.cerevisiae (ScKu70) proteins were aligned using the ClustalW alignment program. Identical residues are marked with black shading, similar residues with grey shading. The position at which the AtKu70 protein is truncated by the T-DNA insertion is indicated with an asterisk. The AtKu70 protein shares 28% identiity with the HsKu70 protein and 16% identity with the ScKu70 protein, respectively.

Figure 2

(A) Genomic organisation of the AtKU70 locus with the insertion point of the T-DNA indicated. The exons of AtKU70 are shown as boxes. The probe used for the DNA blots was a 1871 bp PCR fragment amplified using the primers AK1 + AK3 and is represented as a shaded box. X, XhoI restriction sites. LB, T-DNA left border. The truncated NPTII ORF on the T-DNA is shown as a hatched box. (B) DNA blot. Lane 1, wild-type (Ws) seedlings; lanes 2–5, individual plants containing a T-DNA in AtKU70. (C) Northern analysis: 8 µg total RNA from 12-day-old seedlings was blotted and probed using the 1.8 kb AtKU70 cDNA. Lane 1, Ws; lane 2, AtKU70^–/– seedlings. (D) Integration site of the T-DNA. Upper line, the sequence of exon 10 of the AtKU70 gene is shown in uppercase letters. Intron 10 is indicated in lowercase italics. The sequence deleted due to the T-DNA insertion is in bold. Middle line, the ends of the T-DNA are shown. The left T-DNA border (LB) had lost 9 bp from its end and was integrated into intron 10. The T-DNA right border (RB) end was heavily truncated (∼4 kb lost) resulting in part of the NPTII ORF being fused to AtKU70 exon 10.

Figure 3

(A) MMS sensitivity of AtKU70^–/– plants. Upper row, wild-type plants (ecotype Ws). Lower row, AtKU70^–/– plants. MMS concentrations: i, 0%; ii, 0.006%; iii, 0.008%; iv, 0.01%. (B) X-ray sensitivity of AtKU70^–/– plants. Upper row, wild-type plants (ecotype Ws). Lower row, AtKU70^–/– plants. Seedlings were exposed to: i, 0 Gray; ii, 80 Gray; iii, 100 Gray.

Figure 4

Lengthened telomeres in AtKU70^–/– plants. Lane 1, wild-type (ecotype Ws); lanes 2–7, successive generations of AtKU70^–/– plants. The fragment sizes are shown in kilobase pairs.