Human telomeric protein TRF2 associates with genomic double-strand breaks as an early response to DNA damage.

DNA damage surveillance networks in human cells can activate DNA repair, cell cycle checkpoints and apoptosis in response to fewer than four double-strand breaks (DSBs) per genome. These same networks tolerate telomeres, in part because the protein TRF2 prevents recognition of telomeric ends as DSBs by facilitating their organization into T loops. We now show that TRF2 associates with photo-induced DSBs in nontelomeric DNA in human fibroblasts within 2 s of irradiation. Unlike gammaH2AX, a common marker for DSB damage, TRF2 forms transient foci that colocalize closely with DSBs. The TRF2 DSB response requires the TRF2 basic domain but not its Myb domain and occurs in the absence of functional ATM and DNA-PK protein kinases, MRE11/Rad50/NBS1 complex and Ku70, WRN and BLM repair proteins. Furthermore, overexpression of TRF2 inhibits DSB-induced phosphorylation of ATM signaling targets. Our results implicate TRF2 in an initial stage of DSB recognition and processing that occurs before association of ATM with DSBs and activation of the ATM-dependent DSB response network.

Endogenous hSNM1B/Apollo interacts with TRF2 and stimulates ATM in response to ionizing radiation.

Human SNM1B/Apollo is involved in the cellular response to DNA-damage, however, its precise role is unknown. Recent reports have implicated hSNM1B in the protection of telomeres. We have found hSNM1B to interact with TRF2, a protein which functions in telomere protection and in an early response to ionizing radiation. Here we show that endogenous hSNM1B forms foci which colocalize at telomeres with TRF1 and TRF2. However, we observed that additional hSNM1B foci could be induced upon exposure to ionizing radiation (IR). In live-cell-imaging experiments, hSNM1B localized to photo-induced double-strand breaks (DSBs) within 10s post-induction. Further supporting a role for hSNM1B in the early stages of the cellular response to DSBs, we observed that autophosphorylation of ATM, as well as the phosphorylation of ATM target proteins in response to IR, was attenuated in cells depleted of hSNM1B. These observations suggest an important role for hSNM1B in the response to IR damage, a role that may be, in part, upstream of the central player in maintenance of genome integrity, ATM.

Breakpoints in the ataxia telangiectasia gene arise at the RGYW somatic hypermutation motif.

The mature sporadic T-cell malignancy, T-cell prolymphocytic leukemia (T-PLL) is remarkable for frequently harbouring somatic mutations of the Ataxia Telangiectasia (A-T) gene, ATM. Because some data suggest ATM is frequently rearranged in T-PLL, it was decided to investigate such rearrangements in detail by cloning breakpoints. Among 17 T-PLL tumour samples, three rearrangements were detected by Southern blotting. Two cases harboured a unique type of intragenic duplication in which breakpoints arose at the consensus sequence RGYW/WRCY. The third case harboured a large deletion terminating within the ATM gene. Also, 13 T-cell acute lymphoblastic leukemia (T-ALL) samples were examined and one sample harboured a deletion- insertion with the RGYW motif at the breakpoint in ATM. This is the first known deleterious mutation detected in ATM in T-ALL. Interestingly, the RGYW motif is the signal for a cell-cycle regulated DNA double strand break (DSB) that initiates somatic hypermutation of immunoglobulin and, probably, T-cell receptor genes. The structures of the ATM duplications suggest they may arise from an error in somatic hypermutation. We suggest that aberrant components of somatic hypermutation may contribute to the defective DSB repair characteristic of cancer.

DNA damage-induced phosphorylation of the human telomere-associated protein TRF2.

Several protein kinases from diverse eukaryotes known to perform important roles in DNA repair have also been shown to play critical roles in telomere maintenance. Here, we report that the human telomere-associated protein TRF2 is rapidly phosphorylated in response to DNA damage. We find that the phosphorylated form of TRF2 is not bound to telomeric DNA, as is the ground form of TRF2, and is rapidly localized to damage sites. Our results suggest that the ataxia-telangiectasia-mutated (ATM) protein kinase signal-transduction pathway is primarily responsible for the DNA damage-induced phosphorylation of TRF2. Unlike DNA damage-induced phosphorylation of other ATM targets, the phosphorylated form of TRF2 is transient, being detected rapidly at DNA damage sites postirradiation, but largely dissipated by 2 hours. In addition, we report that the phosphorylated form of TRF2 is present at telomeres in cell types undergoing telomere-based crisis and a recombination-driven, telomerase-independent, alternative lengthening of telomeres (ALT) pathway, likely as a consequence of a telomere-based DNA damage response. Our results link the induction of TRF2 phosphorylation to the DNA damage-response system, providing an example of direct cross-talk via a signaling pathway between these two major cellular processes essential for genomic stability, telomere maintenance, and DNA repair.

ATM mutations are rare in familial chronic lymphocytic leukemia.

It is now recognized that a subset of B-cell chronic lymphocytic leukemia (CLL) is familial. The genetic basis of familial CLL is poorly understood, but recently germ line mutations in the Ataxia Telangiectasia (ATM) gene have been proposed to confer susceptibility to CLL. The evidence for this notion is, however, not unequivocal. To examine this proposition further we have screened the ATM gene for mutations in CLLs from 61 individuals in 29 families. Truncating ATM mutations, including a known ATM mutation, were detected in 2 affected individuals, but the mutations did not cosegregate with CLL in the families. In addition, 3 novel ATM missense mutations were detected. Common ATM missense mutations were not overrepresented. The data support previous observations that ATM mutation is associated with B-CLL. However, ATM mutations do not account for familial clustering of the disease.

The Bloom syndrome helicase BLM interacts with TRF2 in ALT cells and promotes telomeric DNA synthesis.

Telomerase-negative immortalized human cells maintain telomeres by alternative lengthening of telomeres (ALT) pathway(s), which may involve homologous recombination. We find that endogenous BLM protein co-localizes with telomeric foci in ALT human cells but not telomerase positive immortal cell lines or primary cells. BLM interacts in vivo with the telomeric protein TRF2 in ALT cells, as detected by FRET and co-immunoprecipitation. Transient over-expression of green fluorescent protein (GFP)-BLM results in marked, ALT cell-specific increases in telomeric DNA. The association of BLM with telomeres and its effect on telomere DNA synthesis require a functional helicase domain. Our results identify BLM as the first protein found to affect telomeric DNA synthesis exclusively in human ALT cells and suggest that BLM facilitates recombination-driven amplification of telomeres in ALT cells.