Ku acts in a unique way at the mammalian telomere to prevent end joining

Abstract

Telomeres are specialized DNA/protein structures that act as protective caps to prevent end fusion events and to distinguish the chromosome ends from double-strand breaks. We report that TRF1 and Ku form a complex at the telomere. The Ku and TRF1 complex is a specific high-affinity interaction, as demonstrated by several in vitro methods, and exists in human cells as determined by coimmunoprecipitation experiments. Ku does not bind telomeric DNA directly but localizes to telomeric repeats via its interaction with TRF1. Primary mouse embryonic fibroblasts that are deficient for Ku80 accumulated a large percentage of telomere fusions, establishing that Ku plays a critical role in telomere capping in mammalian cells. We propose that Ku localizes to internal regions of the telomere via a high-affinity interaction with TRF1. Therefore, Ku acts in a unique way at the telomere to prevent end joining.

Figure 1

Ku binds TRF1 with high affinity in vitro. (A) Graph of relative response levels obtained for specific TRF1 concentrations after injection was stopped for 10 sec (during dissociation phase) with a best fit curve (Cricket-Graph). RU, Resonance units. (B) Three representative sensorgrams obtained by flowing the indicated concentrations of TRF1 over Ku70/Ku80 covalently attached to the surface of a Biacore CM-5 chip. Curves were aligned on the X- and Y-axes and subtracted against a negative control using BiaEvaluation software. Injection start indicates the point at which TRF1 begins to flow over the Ku-coupled surface, while injection stop indicates the point at which buffer alone begins to flow over the surface. The TRF1 that remained associated with Ku after a 2–3-min dissociation period was removed in subsequent regeneration steps. Injections of each TRF1 concentration were repeated a minimum of three times using two different Ku-coupled CM-5 chips. (C) In vitro translated [³⁵S]methionine labeled His-tagged TRF1 was preincubated with baculovirally expressed purified Ku70/Ku80 heterodimer. The mixtures were immunoprecipitated by p21 antibody (α-p21, lane 1) and by various Ku80 monoclonal antibodies (α-Ku80, lanes 2–5). Immunoprecipitates were resolved on SDS-PAGE followed by autoradiography. The translated His-TRF1 migrated as a 64-kD protein. (C, lower panel) Western analysis of coimmunoprecipitated Ku70 protein by the Ku80 antibodies. (D) Far-Western analysis TRF1 interaction with Ku. BSA (1 μg), TRF1 (200 ng), Ku70/Ku80 (200 ng), and Rad51 (200 ng) proteins were placed on a nitrocellulose membrane, and this membrane was incubated with a ³²P-labeled Ku70/Ku80 probe. (E) PhosphorImager quantitation of ³²P-Ku bound on the membrane. The relative level of Ku bound to these proteins was normalized to the amount of proteins used with BSA set at 1 U.

Figure 2

Ku and TRF1 form a complex in vivo. Cell lysates from HT1080 infected with HA-tagged TRF1 (HA-TRF1) retrovirus or control retrovirus alone (TRF1) were immunoprecipitated with α-Ku80 or α-HA monoclonal antibodies. The immunoprecipitates were resolved by SDS-PAGE and probed with α-Ku80 (A) or α-HA (B).

Figure 3

Telomeric DNA binding analysis by EMSA. (A) Ku does not bind directly to internal telomeric repeats. Binding reactions were performed as described above using a mirocircle containing (TTAGGG)₃ repeats. Lane 1, no protein added; lane 2, 250 nM Ku70/Ku80 only; lane 3, 125 nM TRF1 alone; lane 4, 250 nM Ku was incubated with substrate DNA first, followed by 125 nM TRF1; lane 5, 125 nM TRF1 was incubated with substrate DNA first, followed by 250 nM Ku; lane 6, a preformed complex containing 250 nM Ku and 125 nM TRF1 was incubated with substrate DNA. (B) Microcircles containing random sequence repeats were not bound by both TRF1 and Ku. Reaction mixtures are same as A except that the substrate used was a microcircle containing a random sequence (TAGCAT)₃ repeat. (C) Titration of binding sites. Binding studies were performed using a microcircle containing (TTAGGG)₆ repeats. Lane 1, no protein; lane 2, 125 nM TRF1; lane 3, 250 nM TRF1; lane 4, 500 nM TRF1; lane 5, 250 nM Ku; lane 6, 500 nM Ku. (D) Antibody mediated super-shift. The telomeric microcircle DNA containing (TTAGGG)₃ repeats was incubated with either TRF1 alone (lane 2) or as a preformed complex of TRF1 with Ku70/Ku80 (lanes 3–5,7) as described above, except that the concentration of the poly (dI/dC) was reduced to 0.1 mg/mL. Appropriate antibody was then added to individual reaction mixtures, and the resulting DNA–protein complexes were resolved as described in methods. As a control, mouse IgG_2a was added to one of the reactions (lane 4). Rabbit α-TRF1 antibody (lanes 6,7) or mouse α-Ku70/Ku80 (lane 5) antibodies were used.

Figure 4

FISH analysis of metaphase chromosomal spreads from wild-type and Ku80^−/− mouse embryonic fibroblasts. (A) A metaphase chromosomes from Ku80^+/+ mouse embryonic fibroblast (MEF). Chromosomal DNA was stained with DAPI (blue), and telomeres were hybridized with Cy3-labeled telomere probe (red). (B–C) Chromosome aberrations detected in metaphase spreads from Ku80^−/− MEF. (B) White arrow points to a dicentric chromosome ring; (C) White arrow points to a Robertsonian fusion-like configuration with telomeres at the fusion point. (D) Compilation of the chromosomal analysis of wild-type and Ku80^−/− MEF. Percentages indicate the number of specific chromosomal aberrations per metaphase chromosome set.

Figure 4

FISH analysis of metaphase chromosomal spreads from wild-type and Ku80^−/− mouse embryonic fibroblasts. (A) A metaphase chromosomes from Ku80^+/+ mouse embryonic fibroblast (MEF). Chromosomal DNA was stained with DAPI (blue), and telomeres were hybridized with Cy3-labeled telomere probe (red). (B–C) Chromosome aberrations detected in metaphase spreads from Ku80^−/− MEF. (B) White arrow points to a dicentric chromosome ring; (C) White arrow points to a Robertsonian fusion-like configuration with telomeres at the fusion point. (D) Compilation of the chromosomal analysis of wild-type and Ku80^−/− MEF. Percentages indicate the number of specific chromosomal aberrations per metaphase chromosome set.

Figure 5

Model for Ku localization to the telomere via TRF1. (A) Schematic representation of the 3′ telomeric overhang duplexed within the t-loop (Griffith et al. 1999). Shaded rectangle represents hindrance by specialized t-loop junction proteins such as TRF2. (B) Unwound 3′ telomeric tail during telomeric DNA replication. Shaded oval represents hindrance by telomerase and specialized 3′ overhang telomeric proteins (LaBranche et al. 1998). (C) The TRF1 homodimer bound to internal telomeric tracts with Ku bound to TRF1. Note that Ku is not directly bound to telomeric DNA.