The F-box protein FBX4 targets PIN2/TRF1 for ubiquitin-mediated degradation and regulates telomere maintenance

Pin2/TRF1 was identified previously as both a protein (TRF1) that binds to telomeric DNA repeats and as a protein (Pin2) that associates with the kinase NIMA and suppresses its mitosis inducing activity. Pin2/TRF1 negatively regulates telomere length and also plays a critical role in cell cycle checkpoint control. Pin2/TRF1 is down-regulated in many human cancers and may be degraded by the ubiquitin-proteasome pathway, but components of the pathway involved in Pin2/TRF1 turnover have not been elucidated. By using the two-hybrid system, we recently identified Pin2/TRF1-interacting proteins, PinX1-4, and we demonstrated that PinX1 is a conserved telomerase inhibitor and a putative tumor suppressor. Here we report the characterization of PinX3. PinX3 was later found to be identical to Fbx4, a member of the F-box family of proteins, which function as substrate-specific adaptors of Cul1-based ubiquitin ligases. Fbx4 interacts with both Pin2 and TRF1 isoforms and promotes their ubiquitination in vitro and in vivo. Moreover, overexpression of Fbx4 reduces endogenous Pin2/TRF1 protein levels and causes progressive telomere elongation in human cells. In contrast, inhibition of Fbx4 by RNA interference stabilizes Pin2/TRF1 and promotes telomere shortening, thereby impairing cell growth. These results demonstrate that Fbx4 is a central regulator of Pin2/TRF1 protein abundance and that alterations in the stability of Pin2/TRF1 can have a dramatic impact on telomere length. Thus, Fbx4 may play a critical role in telomere maintenance.     

A specific interaction between the telomeric protein Pin2/TRF1 and the mitotic spindle

Pin2/TRF1 was independently identified as a telomeric DNA binding protein (TRF1) [1] and as a protein (Pin2) that can bind the mitotic kinase NIMA and suppress its ability to induce mitotic catastrophe [2, 3]. Pin2/TRF1 has been shown to bind telomeric DNA as a dimer [3-7] and to negatively regulate telomere length [8-11]. Interestingly, Pin2/TRF1 levels are regulated during the cell cycle, being increased in late G2 and mitosis and degraded as cells exit from mitosis [3]. Furthermore, overexpression of Pin2/TRF1 induces mitotic entry and then apoptosis [12]. This Pin2/TRF1 activity can be significantly potentiated by the microtubule-disrupting agent nocodazole [12] but is suppressed by phosphorylation of Pin2/TRF1 by ATM; this negative regulation is important for preventing apoptosis upon DNA damage [13]. These results suggest a role for Pin2/TRF1 in mitosis. However, nothing is known about how Pin2/TRF1 is involved in mitotic progression. Here, we describe a surprising physical interaction between Pin2/TRF1 and microtubules in a cell cycle-specific manner. Both expressed and endogenous Pin2/TRF1 proteins were localized to the mitotic spindle during mitosis. Furthermore, Pin2/TRF1 directly bound microtubules via its C-terminal domain. Moreover, Pin2/TRF1 also promoted microtubule polymerization in vitro. These results demonstrate for the first time a specific interaction between Pin2/TRF1 and microtubules in a mitosis-specific manner, and they suggest a new role for Pin2/TRF1 in modulating the function of microtubules during mitosis.     

Role of Pin2/TRF1 in telomere maintenance and cell cycle control

Telomeres are specialized structures found at the extreme ends of chromosomes, which have many functions, including preserving genomic stability, maintaining cell proliferative capacity, and blocking the activation of DNA-damage cell cycle checkpoints. Deregulation of telomere length has been implicated in cancer and ageing. Telomere maintenance is tightly regulated by telomerase and many other telomere-associated proteins and is also closely linked to cell cycle control, especially mitotic regulation. However, little is known about the identity and function of the signaling molecules connecting telomere maintenance and cell cycle control. Pin2/TRF1 was originally identified as a protein bound to telomeric DNA (TRF1) and as a protein involved in mitotic regulation (Pin2). Pin2/TRF1 negatively regulates telomere length and importantly, its function is tightly regulated during the cell cycle, acting as an important regulator of mitosis. Recent identification of many Pin2/TRF1 upstream regulators and downstream targets has provided important clues to understanding the dual roles of Pin2/TRF1 in telomere maintenance and cell cycle control. These results have led us to propose that Pin2/TRF1 functions as a key molecule in connecting telomere maintenance and cell cycle control.     

A critical role for Pin2/TRF1 in ATM-dependent regulation. Inhibition of Pin2/TRF1 function complements telomere shortening, radiosensitivity, and the G(2)/M checkpoint defect of ataxia-telangiectasia cells.

Cells derived from patients with the human genetic disorder ataxia-telangiectasia (A-T) display many abnormalities, including telomere shortening, premature senescence, and defects in the activation of S phase and G(2)/M checkpoints in response to double-strand DNA breaks induced by ionizing radiation. We have previously demonstrated that one of the ATM substrates is Pin2/TRF1, a telomeric protein that binds the potent telomerase inhibitor PinX1, negatively regulates telomere elongation, and specifically affects mitotic progression. Following DNA damage, ATM phosphorylates Pin2/TRF1 and suppresses its ability to induce abortive mitosis and apoptosis (Kishi, S., Zhou, X. Z., Nakamura, N., Ziv, Y., Khoo, C., Hill, D. E., Shiloh, Y., and Lu, K. P. (2001) J. Biol. Chem. 276, 29282-29291). However, the functional importance of Pin2/TRF1 in mediating ATM-dependent regulation remains to be established. To address this question, we directly inhibited the function of endogenous Pin2/TRF1 in A-T cells by stable expression of two different dominant-negative Pin2/TRF1 mutants and then examined their effects on telomere length and DNA damage response. Both the Pin2/TRF1 mutants increased telomere length in A-T cells, as shown in other cells. Surprisingly, both the Pin2/TRF1 mutants reduced radiosensitivity and complemented the G(2)/M checkpoint defect without inhibiting Cdc2 activity in A-T cells. In contrast, neither of the Pin2/TRF1 mutants corrected the S phase checkpoint defect in the same cells. These results indicate that inhibition of Pin2/TRF1 in A-T cells is able to bypass the requirement for ATM in specifically restoring telomere shortening, the G(2)/M checkpoint defect, and radiosensitivity and demonstrate a critical role for Pin2/TRF1 in the ATM-dependent regulation of telomeres and DNA damage response.     

C-terminal amino acids 290-328 of LPTS/PinX1 confer telomerase inhibition

LPTS/PinX1, a telomerase inhibitor composed of 328 amino acids, binds to the telomere associated protein Pin2/TRF1 and to the telomerase catalytic subunit hTERT. However, the mechanism by which LPTS/PinX1 regulates telomerase activity remains unclear. Here we show, for the first time, that LPTS/PinX1 uses different domains to interact with Pin2/TRF1 and hTERT. The LPTS/PinX1_254-289 fragment specifically binds to Pin2/TRF1, and LPTS/PinX1_290-328 can associate with hTERT. Compared with the full-length LPTS/PinX1 protein, LPTS/PinX1_290-328 shows stronger in vitro telomerase inhibitory activity. Moreover, the LPTS/PinX1 protein was recruited to telomeres for binding to Pin2/TRF1. Overexpression of LPTS/PinX1_290-328, which contains a nucleolus localization signal, in cells resulted in telomere shortening and progressive cell death. Conversely, telomere elongation was induced by expression of the dominant-negative LPTS/PinX1_1-289. Our results suggest that the C-terminal fragment of LPTS/PinX1 (LPTS/PinX1_290-328) contains a telomerase inhibitory domain that is required for the inhibition of telomere elongation and the induction of cell crisis. Our studies also provide evidence that LPTS/PinX1 interaction with Pin2/TRF1 may play a role in the stabilization of telomeres.     

Involvement of the telomeric protein Pin2/TRF1 in the regulation of the mitotic spindle

Pin2/TRF1 was independently identified as a telomeric DNA-binding protein (TRF1) that regulates telomere length, and as a protein (Pin2) that can bind the mitotic kinase NIMA and suppress its lethal phenotype. We have previously demonstrated that Pin2/TRF1 levels are cell cycle-regulated and its overexpression induces mitotic arrest and then apoptosis. This Pin2/TRF1 activity can be potentiated by microtubule-disrupting agents, but suppressed by phosphorylation of Pin2/TRF1 by ATM; this negative regulation is critical in mediating for many, but not all, ATM-dependent phenotypes. Interestingly, Pin2/TRF1 specifically localizes to mitotic spindles in mitotic cells and affects the microtubule polymerization in vitro. These results suggest a role of Pin2/TRF1 in mitosis. However, nothing is known about whether Pin2/TRF1 affects the spindle function in mitotic progression. Here we characterized a new Pin2/TRF1-interacting protein, EB1, that was originally identified in our yeast two-hybrid screen. Pin2/TRF1 bound EB1 both in vitro and in vivo and they also co-localize at the mitotic spindle in cells. Furthermore, EB1 inhibits the ability of Pin2/TRF1 to promote microtubule polymerization in vitro. Given that EB1 is a microtubule plus end-binding protein, these results further confirm a specific interaction between Pin2/TRF1 and the mitotic spindle. More importantly, we have shown that inhibition of Pin2/TRF1 in ataxia-telangiectasia cells is able to fully restore their mitotic spindle defect in response to microtubule disruption, demonstrating for the first time a functional involvement of Pin2/TRF1 in mitotic spindle regulation.     

Telomeric protein Pin2/TRF1 as an important ATM target in response to double strand DNA breaks

ATM mutations are responsible for the genetic disease ataxia-telangiectasia (A-T). ATM encodes a protein kinase that is activated by ionizing radiation-induced double strand DNA breaks. Cells derived from A-T patients show many abnormalities, including accelerated telomere loss and hypersensitivity to ionizing radiation; they enter into mitosis and apoptosis after DNA damage. Pin2 was originally identified as a protein involved in G(2)/M regulation and is almost identical to TRF1, a telomeric protein that negatively regulates telomere elongation. Pin2 and TRF1, probably encoded by the same gene, PIN2/TRF1, are regulated during the cell cycle. Furthermore, up-regulation of Pin2 or TRF1 induces mitotic entry and apoptosis, a phenotype similar to that of A-T cells after DNA damage. These results suggest that ATM may regulate the function of Pin2/TRF1, but their exact relationship remains unknown. Here we show that Pin2/TRF1 coimmunoprecipitated with ATM, and its phosphorylation was increased in an ATM-dependent manner by ionizing DNA damage. Furthermore, activated ATM directly phosphorylated Pin2/TRF1 preferentially on the conserved Ser(219)-Gln site in vitro and in vivo. The biological significance of this phosphorylation is substantiated by functional analyses of the phosphorylation site mutants. Although expression of Pin2 and its mutants has no detectable effect on telomere length in transient transfection, a Pin2 mutant refractory to ATM phosphorylation on Ser(219) potently induces mitotic entry and apoptosis and increases radiation hypersensitivity of A-T cells. In contrast, Pin2 mutants mimicking ATM phosphorylation on Ser(219) completely fail to induce apoptosis and also reduce radiation hypersensitivity of A-T cells. Interestingly, the phenotype of the phosphorylation-mimicking mutants is the same as that which resulted from inhibition of endogenous Pin2/TRF1 in A-T cells by its dominant-negative mutants. These results demonstrate for the first time that ATM interacts with and phosphorylates Pin2/TRF1 and suggest that Pin2/TRF1 may be involved in the cellular response to double strand DNA breaks.     

Telomerase inhibitor PinX1 provides a link between TRF1 and telomerase to prevent telomere elongation

Telomere maintenance is essential for protecting chromosome ends. Aberrations in telomere length have been implicated in cancer and aging. Telomere elongation by human telomerase is inhibited in cis by the telomeric protein TRF1 and its associated proteins. However, the link between TRF1 and inhibition of telomerase elongation of telomeres remains elusive because TRF1 has no direct effect on telomerase activity. We have previously identified one Pin2/TRF1-interacting protein, PinX1, that has the unique property of directly binding and inhibiting telomerase catalytic activity (Zhou, X. Z., and Lu, K. P. (2001) Cell 107, 347-359). However, nothing is known about the role of the PinX1-TRF1 interaction in the regulation of telomere maintenance. By identifying functional domains and key amino acid residues in PinX1 and TRF1 responsible for the PinX1-TRF1 interaction, we show that the TRF homology domain of TRF1 interacts with a minimal 20-amino acid sequence of PinX1 via hydrophilic and hydrophobic interactions. Significantly, either disrupting this interaction by mutating the critical Leu-291 residue in PinX1 or knocking down endogenous TRF1 by RNAi abolishes the ability of PinX1 to localize to telomeres and to inhibit telomere elongation in cells even though neither has any effect on telomerase activity per se. Thus, the telomerase inhibitor PinX1 is recruited to telomeres by TRF1 and provides a critical link between TRF1 and telomerase inhibition to prevent telomere elongation and help maintain telomere homeostasis.     

Functional subdomain in the ankyrin domain of tankyrase 1 required for poly(ADP-ribosyl)ation of TRF1 and telomere elongation

In human cells, telomere elongation by telomerase is repressed in cis by the telomeric protein TRF1. Tankyrase 1 binds TRF1 via its ankyrin domain and poly(ADP-ribosyl)ates it. Overexpression of tankyrase 1 in telomerase-positive cells releases TRF1 from telomeres, resulting in telomere elongation. The tankyrase 1 ankyrin domain is classified into five conserved subdomains, ARCs (ankyrin repeat clusters) I to V. Here, we investigated the biological significance of the ARCs. First, each ARC worked as an independent binding site for TRF1. Second, ARCs II to V recognized the N-terminal acidic domain of TRF1 whereas ARC I bound a discrete site between the homodimerization and the Myb-like domains of TRF1. Inactivation of TRF1 binding in the C-terminal ARC, ARC V, either by deletion or point mutation, significantly reduced the ability of tankyrase 1 to poly(ADP-ribosyl)ate TRF1, release TRF1 from telomeres, and elongate telomeres. In contrast, other ARCs, ARC II and/or IV, inactivated by point mutations still retained the biological function of tankyrase 1. On the other hand, ARC V per se was not sufficient for telomere elongation, suggesting a structural role for multiple ARCs. This work provides evidence that specific ARC-TRF1 interactions play roles in the essential catalytic function of tankyrase 1.     

Targeting assay to study the cis functions of human telomeric proteins: evidence for inhibition of telomerase by TRF1 and for activation of telomere degradation by TRF2

We investigated the control of telomere length by the human telomeric proteins TRF1 and TRF2. To this end, we established telomerase-positive cell lines in which the targeting of these telomeric proteins to specific telomeres could be induced. We demonstrate that their targeting leads to telomere shortening. This indicates that these proteins act in cis to repress telomere elongation. Inhibition of telomerase activity by a modified oligonucleotide did not further increase the pace of telomere erosion caused by TRF1 targeting, suggesting that telomerase itself is the target of TRF1 regulation. In contrast, TRF2 targeting and telomerase inhibition have additive effects. The possibility that TRF2 can activate a telomeric degradation pathway was directly tested in human primary cells that do not express telomerase. In these cells, overexpression of full-length TRF2 leads to an increased rate of telomere shortening.     

Dynamics of protein binding to telomeres in living cells: implications for telomere structure and function

Telomeric proteins have an essential role in the regulation of the length of the telomeric DNA tract and in protection against end-to-end chromosome fusion. Telomere organization and how individual proteins are involved in different telomere functions in living cells is largely unknown. By using green fluorescent protein tagging and photobleaching, we investigated in vivo interactions of human telomeric DNA-binding proteins with telomeric DNA. Our results show that telomeric proteins interact with telomeres in a complex dynamic fashion: TRF2, which has a dual role in chromosome end protection and telomere length homeostasis, resides at telomeres in two distinct pools. One fraction ( approximately 73%) has binding dynamics similar to TRF1 (residence time of approximately 44 s). Interestingly, the other fraction of TRF2 binds with similar dynamics as the putative end-protecting factor hPOT1 (residence time of approximately 11 min). Our data support a dynamic model of telomeres in which chromosome end-protection and telomere length homeostasis are governed by differential binding of telomeric proteins to telomeric DNA.     

Functional interaction between poly(ADP-Ribose) polymerase 2 (PARP-2) and TRF2: PARP activity negatively regulates TRF2

The DNA damage-dependent poly(ADP-ribose) polymerase-2 (PARP-2) is, together with PARP-1, an active player of the base excision repair process, thus defining its key role in genome surveillance and protection. Telomeres are specialized DNA-protein structures that protect chromosome ends from being recognized and processed as DNA strand breaks. In mammals, telomere protection depends on the T(2)AG(3) repeat binding protein TRF2, which has been shown to remodel telomeres into large duplex loops (t-loops). In this work we show that PARP-2 physically binds to TRF2 with high affinity. The association of both proteins requires the N-terminal domain of PARP-2 and the myb domain of TRF2. Both partners colocalize at promyelocytic leukemia bodies in immortalized telomerase-negative cells. In addition, our data show that PARP activity regulates the DNA binding activity of TRF2 via both a covalent heteromodification of the dimerization domain of TRF2 and a noncovalent binding of poly(ADP-ribose) to the myb domain of TRF2. PARP-2(-/-) primary cells show normal telomere length as well as normal telomerase activity compared to wild-type cells but display a spontaneously increased frequency of chromosome and chromatid breaks and of ends lacking detectable T(2)AG(3) repeats. Altogether, these results suggest a functional role of PARP-2 activity in the maintenance of telomere integrity.     

Topoisomerase IIIalpha is required for normal proliferation and telomere stability in alternative lengthening of telomeres

Topoisomerase (Topo) IIIalpha associates with BLM helicase, which is proposed to be important in the alternative lengthening of telomeres (ALT) pathway that allows telomere recombination in the absence of telomerase. Here, we show that human Topo IIIalpha colocalizes with telomeric proteins at ALT-associated promyelocytic bodies from ALT cells. In these cells, Topo IIIalpha immunoprecipitated with telomere binding protein (TRF) 2 and BLM and was shown to be associated with telomeric DNA by chromatin immunoprecipitation, suggesting that these proteins form a complex at telomere sequences. Topo IIIalpha depletion by small interfering RNA reduced ALT cell survival, but did not affect telomerase-positive cell lines. Moreover, repression of Topo IIIalpha expression in ALT cells reduced the levels of TRF2 and BLM proteins, provoked a strong increase in the formation of anaphase bridges, induced the degradation of the G-overhang signal, and resulted in the appearance of DNA damage at telomeres. In contrast, telomere maintenance and TRF2 levels were unaffected in telomerase-positive cells. We conclude that Topo IIIalpha is an important telomere-associated factor, essential for telomere maintenance and chromosome stability in ALT cells, and speculate on its potential mechanistic function.     

Regulation of telomeric repeat binding factor 1 binding to telomeres by casein kinase 2-mediated phosphorylation

Telomere maintenance is essential for continued cell proliferation and chromosome stability. Telomeres are maintained by telomerase and a collection of associated proteins. The telomeric protein telomeric repeat binding factor 1 (TRF1) negatively regulates telomere length by inhibiting access of telomerase at telomere termini. Here we report that TRF1 interacts with the beta subunit of casein kinase 2 (CK2) and serves as a substrate for CK2. CK2-mediated phosphorylation is required for the efficient telomere binding of TRF1 in vitro and in vivo. Inhibition of CK2 by the CK2 inhibitor 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole decreased the ability of TRF1 to bind telomeric DNA. The resulting telomere-unbound form of TRF1 was then ubiquitinated and degraded by the proteasome. Partial knockdown of CK2 by small interfering RNA resulted in removal of TRF1 from telomeres and subsequent degradation of TRF1. Mapping of the CK2 target site identified threonine 122 as a substrate in TRF1. A threonine to alanine change at this position led to a diminished DNA binding due to reduced dimerization of TRF1. In addition, phosphorylation of threonine 122 seemed critical for TRF1-mediated telomere length control. Our findings suggest that CK2-mediated phosphorylation of TRF1 plays an important role in modulating telomere length homeostasis by determining the levels of TRF1 at telomeres.     

XPF with mutations in its conserved nuclease domain is defective in DNA repair but functions in TRF2-mediated telomere shortening

TRF2, a telomere-binding protein, is a crucial player in telomere length maintenance. Overexpression of TRF2 results in telomere shortening in both normal primary fibroblasts and telomerase-positive cancer cells. TRF2 is found to be associated with XPF-ERCC1, a structure-specific endonuclease involved in nucleotide excision repair, crosslink repair and DNA recombination. XPF-ERCC1 is implicated in TRF2-dependent telomere loss in mouse keratinocytes, however, whether XPF-ERCC1 and its nuclease activity are required for TRF2-mediated telomere shortening in human cells is unknown. Here we report that TRF2-induced telomere shortening is abrogated in human cells deficient in XPF, demonstrating that XPF-ERCC1 is required for TRF2-promoted telomere shortening. To further understand the role of XPF in TRF2-dependent telomere shortening, we generated constructs containing either wild type XPF or mutant XPF proteins carrying amino acid substitutions in its conserved nuclease domain. We show that wild type XPF can complement XPF-deficient cells for repair of UV-induced DNA damage whereas the nuclease-inactive XPF proteins fail to do so, indicating that the nuclease activity of XPF is essential for nucleotide excision repair. In contrast, both wild type XPF and nuclease-inactive XPF proteins, when expressed in XPF-deficient cells, are able to rescue TRF2-mediated telomere shortening. Thus, our results suggest that the function of XPF in TRF2-mediated telomere shortening is conserved between mouse and human. Furthermore, our findings reveal an unanticipated nuclease-independent function of XPF in TRF2-mediated telomere shortening.     

Control of human telomere length by TRF1 and TRF2

Telomere length in human cells is controlled by a homeostasis mechanism that involves telomerase and the negative regulator of telomere length, TRF1 (TTAGGG repeat binding factor 1). Here we report that TRF2, a TRF1-related protein previously implicated in protection of chromosome ends, is a second negative regulator of telomere length. Overexpression of TRF2 results in the progressive shortening of telomere length, similar to the phenotype observed with TRF1. However, while induction of TRF1 could be maintained over more than 300 population doublings and resulted in stable, short telomeres, the expression of exogenous TRF2 was extinguished and the telomeres eventually regained their original length. Consistent with their role in measuring telomere length, indirect immunofluorescence indicated that both TRF1 and TRF2 bind to duplex telomeric DNA in vivo and are more abundant on telomeres with long TTAGGG repeat tracts. Neither TRF1 nor TRF2 affected the expression level of telomerase. Furthermore, the presence of TRF1 or TRF2 on a short linear telomerase substrate did not inhibit the enzymatic activity of telomerase in vitro. These findings are consistent with the recently proposed t loop model of telomere length homeostasis in which telomerase-dependent telomere elongation is blocked by sequestration of the 3' telomere terminus in TRF1- and TRF2-induced telomeric loops.     

人端粒结合蛋白TRF1的克隆、表达和抗体制备

利用RT-PCR技术,从HeLa细胞的cDNA文库中扩增到人端粒结合蛋白1(hTRF1)基因编码区序列,克隆至pUCm-T载体,测序正确后,构建带His₆-tag原核表达载体pET-28c-TRF1,经IPTG诱导表达的His₆-TRF1融合蛋白分子量约为65kD,Western-blot证实表达产物可特异地与TRF1抗体sc-6165结合。用Ni^2＋NTA胶亲和层析纯化可得到电泳均一的融合蛋白,免疫新西兰纯种大白兔,获得特异性好的多克隆抗体,该抗体可用于免疫荧光染色和Western-blot方法检测哺乳动物细胞内源性的TRF1分子。     

端粒结合蛋白TRF2的研究进展

端粒DNA结合蛋白TRF2(TTAGGG repeat binding factor-2)以二聚体形式通过Myb结构域与端粒重复序列TTAGGG结合,并与TRF1、TIN2、Rap1、TINT1及POT1蛋白组成Shelterin蛋白复合物,协同在端粒动态平衡维持过程中起关键作用,进而影响整个基因组的稳定性。此外,TRF2在细胞DNA损伤应答过程中可能发挥重要作用。本文将对TRF2结构和功能研究的最新进展进行综述。     

Unwinding protein complexes in ALTernative telomere maintenance

Telomeres are composed of specialized chromatin that includes DNA repair/recombination proteins, telomere DNA‐binding proteins and a number of three dimensional nucleic acid structures including G‐quartets and D‐loops. A number of studies suggest that the BLM and WRN recQ‐like helicases play important roles in recombination‐mediated mechanisms of telomere elongation or A lternative L engthening of T elomeres (ALT), processes that maintain/elongate telomeres in the absence of telomerase. BLM and WRN localize within ALT‐associated nuclear bodies in telomerase‐negative immortalized cell lines and interact with the telomere‐specific proteins POT1, TRF1 and TRF2. Helicase activity is modulated by these interactions. BLM functions in DNA double‐strand break repair processes such as non‐homologous end joining, homologous recombination‐mediated repair, resolution of stalled replication forks and synthesis‐dependent strand annealing, although its precise functions at the telomeres are speculative. WRN also functions in DNA replication, recombination and repair, and in addition to its helicase domain, includes an exonuclease domain not found in other recQ‐like helicases. The biochemical properties of BLM and WRN are, therefore, important in biological processes other than DNA replication, recombination and repair. In this review, we discuss some previous and recent findings of human rec‐Q‐like helicases and their role in telomere elongation during ALT processes. J. Cell. Biochem. 109: 7–15, 2010. © 2009 Wiley‐Liss, Inc.