Effects of double-strand break repair proteins on vertebrate telomere structure

Abstract

Although telomeres are not recognized as double-strand breaks (DSBs), some DSB repair proteins are present at telomeres and are required for telomere maintenance. To learn more about the telomeric function of proteins from the homologous recombination (HR) and non-homologous end joining pathways (NHEJ), we have screened a panel of chicken DT40 knockout cell lines for changes in telomere structure. In contrast to what has been observed in Ku-deficient mice, we found that Ku70 disruption did not result in telomere-telomere fusions and had no effect on telomere length or the structure of the telomeric G-strand overhang. G-overhang length was increased by Rad51 disruption but unchanged by disruption of DNA-PKcs, Mre11, Rad52, Rad54, XRCC2 or XRCC3. The effect of Rad51 depletion was unexpected because gross alterations in telomere structure have not been detected in yeast HR mutants. Thus, our results indicate that Rad51 has a previously undiscovered function at vertebrate telomeres. They also indicate that Mre11 is not required to generate G-overhangs. Although Mre11 has been implicated in overhang generation, overhang structure had not previously been examined in Mre11-deficient cells. Overall our findings indicate that there are significant species-specific differences in the telomeric function of DSB repair proteins.

Figure 1

Proteins involved in DSB repair in vertebrate cells. During NHEJ the DNA-PK complex, which consists of the Ku70/Ku80 heterodimer and the DNA-PK catalytic subunit, binds the broken ends and facilitates rejoining by DNA ligase IV and XRCC4. HR is carried out by proteins in the Rad52 epistasis group. These include Rad51, Rad52, Rad54 and the Rad51 paralogs Rad51B, Rad51C, Rad51D, XRCC2, XRCC3, Rad50, Mre11 and Nbs1. During the repair process, Rad51 aided by other accessory factors catalyzes strand exchange with a homologous DNA molecule. The Rad50/Mre11/Nbs1 complex, which has nuclease and DNA-unwinding activities, participates in both HR and NHEJ in ways that are still not fully understood.

Figure 2

Isolation of Ku70⁺ and Ku70^–/– clones. (A) Scheme for isolating individual Ku70 clones. (B) Western blot showing expression of FcKu70 in Ku^–/– cells transfected with FcKu70 cDNA. Lane 1, wild-type DT40; lanes 2–6, Ku⁺ TelL clones; lanes 7 and 8, Ku70^–/– cells. The arrow marks the position of Ku70 and FcKu70; the arrowhead marks a cross-reacting band that was used to normalize for the amount of protein loaded in each lane. The level of FcKu70 expression relative to wild-type cKu70 expression is given below each lane. (C) Rescue of γ-radiation sensitivity by FcKu70 expression. Cells were treated with 2 Gy γ-radiation at the indicated times following release from nocodazole arrest. The fraction of colonies surviving compared with non-irradiated controls of the same genotype are shown for the 5 h time point. Ku70⁺-1 and Ku70⁺-2 represent two different Ku⁺ TelL clones.

Figure 3

Effect of Ku70 deficiency on telomere length. (A) Telomere length analysis of Ku70^–/– and Ku70⁺ TelL clones. Telomeric restriction fragments were identified by in-gel hybridization to ³²P-labeled (C₃TA₂)₄ probe and the weighted mean telomere length was calculated following quantification of the hybridization signal by PhosphorImager. Size (in kb) of DNA markers is shown to the left. (B) Histogram showing telomere lengths in Ku70^–/– and Ku70⁺ TelL clones. Column 1, telomere length of parental Ku70^–/– TelL clone at PD0; column 2, average telomere length at PD50 of five Ku70⁺ TelL clones; column 3, average telomere length at PD50 of five Ku70^–/– TelL clones. (C) Histogram showing telomere lengths in Ku70^–/– and Ku70⁺ TelS clones. Column 1, average telomere length of parental Ku70^–/– TelS clone at PD0; column 2, average telomere length at PD50 of three Ku70⁺ TelS clones; column 3, average telomere length at PD50 of three Ku70^–/– TelS clones.

Figure 4

Representative metaphase spreads from wild-type and Ku70^–/– cells. Chromosomes were Giemsa stained. Note the lack of end-to-end fusions in the Ku^–/– cells. (A) Wild-type cells. (B) Ku70^–/–.

Figure 5

Comparison of G-strand overhang length by in-gel hybridization. The upper panels show in-gel hybridization of ³²P-labeled (C₃AT₂)₄ probe to genomic DNA from DT40 cells deficient for various DSB repair proteins. The lower panels show loading controls. The bottom portion of each gel was removed before the in-gel hybridization, denatured, blotted to membrane and hybridized with a probe for total genomic DNA. (A) Ku70-, XRCC2-, XRCC3- and DNA-PKcs-deficient cell lines. As none of these cell lines contained a conditional allele, the hybridization signal for each mutant cell line was compared with the signal from wild-type samples run on the same gel (lanes 7 and 8). Lanes 9 and 10, and 11 and 12, contain samples from two different Ku70^–/– clones with long telomeres. The total amount of G-overhang signal (×10^–6) from the PhosphorImager analysis is given at the bottom of the upper panel while the total signal from the loading control is given at the bottom of the lower panel. The relative G-overhang signal for each lane is given as a percentage of the wild-type signal. (B) Rad51 conditional cells. Lanes 1 and 2, cells isolated at PD0; lanes 3 and 4, cells grown without doxycycline isolated at PD4; lanes 5 and 6, cells grown with doxycycline isolated at PD4. (C) Mre11 conditional cells. Lanes 1 and 2, cells isolated at PD0; lanes 3 and 4, cells grown without doxycycline isolated at PD7; lanes 5 and 6, cells grown with doxycycline isolated at PD7.