WRN Controls Formation of Extrachromosomal Telomeric Circles and Is Required for TRF2^ΔB-Mediated Telomere Shortening

Abstract

Telomere dysfunction has been proposed to contribute to the pathogenesis of Werner syndrome (WS), a premature-aging disorder. The WS protein WRN binds TRF2, a telomere-specific factor that protects chromosome ends. TRF2 possesses an amino-terminal domain that plays an essential role in preventing telomere shortening, as expression of TRF2 ⌬B , which lacks this domain, leads to the formation of telomeric circles, telomere shortening, and cell senescence. Our data show that the TRF2 ⌬B -induced telomeric-loop homologous-recombination pathway requires WRN helicase. In addition, we show that WRN represses the formation of spontaneous telomeric circles, as demonstrated by the increased levels of telomeric circles observed in telomerase-positive WS fibroblasts. The mechanism of circle formation in WS cells does not involve XRCC3 function. Circle formation in WS cells is reduced by reconstitution with wild-type WRN but not mutant forms lacking either exonuclease or helicase activity, demonstrating that both enzymatic activities of WRN are required to suppress telomeric-circle formation in normal cells expressing telomerase reverse transcriptase. Thus, WRN has a key protective function at telomeres which influences telomere topology and inhibits accelerated attrition of telomeres.Werner syndrome (WS) is an autosomal recessive disease that is characterized by accelerated aging and the premature onset of several age-associated disorders such as cardiovascular disease and cancer (11). At the cellular level, WS cells show a significant level of genome instability and hypersensitivity to genotoxic agents such as camptothecin, 4-nitroquinoline-Noxide, and mitomycin C (12, 24, 28-31). The disease is caused by loss-of-function mutations in the gene that encodes a member of the RecQ family of helicases (RecQ3 or WS protein WRN) (35). WRN, in contrast to other RecQ helicases, possesses a unique amino-terminal domain with exonuclease activity. The presence of both exonuclease and helicase activities indicates that WRN may be involved in the processing of double-stranded DNA molecules, and identification of proteins that interact with WRN has further suggested that this protein may have a function in DNA damage repair, replication, or recombination (27).All of the physiological substrates for WRN have yet to be identified; however, one bona fide substrate for WRN is telomeric DNA (27). Human telomeres are composed of several kilobases of the repetitive hexamer TTAGGG and contain a 3Ј single-stranded DNA extension that is thought to loop back and invade the proximal complementary strand, thereby leading to the establishment of a protective structure termed the telomeric loop (t-loop) (13, 23). The formation and maintenance of the t-loop are mediated by a multiprotein complex which includes telomere-specific proteins such as telomere repeat factor 1 (TRF1) and TRF2 that bind telomeres through a myb-like DNA binding domain located at the carboxy-terminal end (1, 3, 4). These proteins are believed to protect telomeres from end-to-end f...

Ku86 represses lethal telomere deletion events in human somatic cells

“…TRD is inhibited by Ku (as is the telomere loss we have described here), and it requires HR activities. In this light, it is important to note that the formation of t-circles in human ALT cells requires the HR genes XRCC3 and NBS1 (34,35) whereas the t-circles observed in WRN patient cells (30) and the telomere shortening events observed in plants (32) do not. This suggests that there are at least 2 discrete mechanisms for t-circle formation, only one of which parallels yeast TRD.…”

Shelterin complex and associated factors at human telomeres

“…Therefore, WS cells were analyzed for t-circles formation, and indeed these cells showed a significant increase in these structures, which was reduced upon re-introduction of the WRN cDNA, but not with a helicase-defective mutant. 100 Thus, it appears that recruitment of WRN is important for the suppression of intra-telomere recombination and subsequent t-circle formation. Such an increase in t-circle formation has been documented in ALT cells.…”

“…TRF2 can also be phosphorylated or sumoylated, and its telomeric association strongly increases in S phase. 100 However, a great deal of the impact of shelterin resides in the transient or partial recruitment of a number of molecules which are not quantitatively at telomeres, not present there throughout the cell cycle and may have known established functions elsewhere in the cell. These telomere-associated factors establish in specific cases groups of activities, which, together, mediate telomere function in chromosome stability, or allow for efficient telomere processing and passage through the S and M phases of the cell cycle.…”

Ku's essential role in keeping telomeres intact

“…This domain is required for TRF2 inhibition of resolvase cleavage of telomeric HJs in vitro and both mouse and human cells expressing high levels of TRF2⌬B, which lacks the domain, exhibit high levels of t-circles (9, 10). Additional factors implicated in t-circle formation in response to TRF2⌬B expression include XRCC3, which is associated with HJ resolvase activity in mammalian cells; Nbs1, a component of the Mre11 complex, which is involved in recombinational repair; and WRN, an helicase/ exonuclease, which interacts directly with TRF2 (9,11).…”