telomeres , unique structures at the physical ends of linear eukaryotic chromosomes , were first described almost 70 yearsago by hermann muller in his classic studies of the fruit fly drosophilia melanogaster ( 1 ) . he noted that chromosomal inversions resulting from ionizing radiation ( ir)-induced double - strand breaks ( dsbs ) never involved the very end of a chromosome rejoining with some other part of a chromosome . telomere , which comes from greek  telos meaning end and meros meaning part  based on this chromosome end protection phenomenon . shortly thereafter , barbara mcclintock observed that while broken ends of maize chromosome fused , forming dicentric chromosomes , unbroken chromosomes rarely fused ; i.e. natural chromosomal termini are not the absence of interstitial telomere sequence within ir - induced dicentrics was later verified in human cells ( 3 ) . these studies demonstrate that normally cells accurately distinguish telomeric ends from random dsb ends and protect the former from illegitimate end - joining reactions . how cells make this critical distinction continues to be an active area of research today , especially as the dividing lines between the two types of ends have become less , rather than more clear . recent discoveries that certain dsb repair proteins act to preserve  rather than to join  the natural ends of mammalian chromosomes ( 47 ) , have provided impetus for the union of two seemingly disparate scientific fields , dna repair and telomere biology . here , we focus on the creation of dysfunctional mammalian telomeres in various repair deficient backgrounds that result from either the loss of end - capping structure or the loss of terminal sequence ( shortening ) , and the consequences of this loss of function . telomeres serve multiple functions in preserving chromosome stability , including protecting the ends of chromosomes from degradation and preventing chromosomal end fusion . the dna component of telomeres consists of tandem arrays of short , repetitive g - rich sequence [ ttaggg in vertebrates , ( 8,9 ) ] , oriented 5-to-3 towards the end of the chromosome ( 10 ) , ending in an essential 3 single - stranded overhang that ranges in length from 50 to 400 nt ( 1113 ) . electron microscopy studies suggest this overhang can loop back and integrate into the duplex repeat tract , forming a t - loop ( 14 ) , an attractive , although not necessarily exclusive , architectural solution to the end - capping dilemma . telomeres gradually shorten in replicating cells due to end - processing and the  end replication problem ( 1518 ) , so in order for continuous cell division to occur , they must be replenished . the addition of telomeric repeats de novo is accomplished by the reverse transcriptase telomerase ( 19,20 ) . thus , telomere length maintenance is a state of equilibrium between telomere loss and re - addition . precisely how telomere length regulation occurs is unknown , but it does appear that the shortest telomeres are preferentially targeted for elongation by telomerase ( 21 ) . telomere length is maintained in germ line cells , however , most human somatic cells do not express sufficient telomerase activity to prevent telomere loss as they divide . as a result , telomeres eventually shorten to the point where they initiate a cell cycle arrest in g1 , a state termed replicative senescence ( 22 ) . telomere shortening can , therefore , limit the number of times somatic cells divide , contributing not only to aging phenotypes , but also providing an effective tumor suppressor mechanism . cells that lose the ability to senesce because of mutations in p53 protein continue to divide , eventually entering , cells that constitutively express telomerase can continue to divide almost indefinitely ( 23,24 ) . consistent with the requirement for telomere maintenance as a step in carcinogenesis , most tumor cells express telomerase ( 25 ) . however , an alternative mechanism for telomere maintenance has also been described ( 26,27 ) , which is observed in some tumors ( 28 ) and involves recombination ( 26,29 ) . although telomerase is responsible for addition of telomeric sequence with every cell cycle , a plethora of other proteins also play important roles in the regulation of telomere length maintenance and in the formation of a protective end - cap that prevents chromosome fusion ( 30,31 ) . proteins that directly bind the double - stranded telomeric repeats include the ttaggg repeat factors trf1 ( 32,33 ) and trf2 ( 34,35 ) . pot1 ( protection of telomeres 1 ) specifically recognizes telomeric single - stranded dna , belongs to a family of oligosaccharide / oligonucleotide - binding ( ob)-fold - containing proteins and is highly conserved among eukaryotes ( 36,37 ) . telomere - associated proteins that do not bind dna directly include tin2 ( trf1-interacting nuclear protein 2 ) , which associates with trf1 ( 38 ) and trf2 ( 39,40 ) . the trf1 complex contains both tin2 and pot1 and acts to regulate telomere - length homeostasis . tpp1 , the recently proposed name ( 31 ) for a pot1-interacting protein identified independently in three laboratories [ tint1 ( 41 ) , ptop ( 42 ) and pip1 ( 43 ) ] , recruits pot1 to telomeres and so is also involved in telomere length regulation . rap1 ( human repressor activator protein ) ( 44 ) is recruited to telomeres by trf2 and has been shown to negatively regulate telomere length in vivo ( 45 ) . six proteins have been proposed to form an essential , dynamic complex at human telomeres : trf1 , trf2 , pot1 , tin2 , tpp1 and rap1 . this mammalian telomeric core complex serves to form and protect the telomere , and has been termed both the telosome ( 42 ) and alternately , shelterin ( 31 ) . other proteins , many of which are more commonly associated with dna repair , are also found at telomeric ends [ reviewed in ( 46 ) ] . examples include dna - pk ( ku70/ku86/dna - pkcs ) , the mrn complex ( mre11/rad50/nbs1 ) , parp1/2 , tankyrase 1/2 , atm , ercc1/xpf , rad51d , wrn and blm . trf2 has been shown to bind to atm , blocking a damage response at telomeres ( 47 ) . interestingly , it has recently been proposed that normally , functional human telomeres must be recognized as dna damage in the g2 phase of the cell cycle , in order to recruit the processing machinery necessary for formation of a functional telomere ( 48 ) . the interplay between telomeres and dsbs may be better understood not by viewing the striking differences between them , but instead by viewing the obvious similarity  both are dna ends , the very substrate telomere and damage response / repair proteins specifically recognize and bind . functional telomeres are essential for continuous cellular proliferation , and therefore loss of chromosomal end - capping has consequences in both aging and carcinogenesis ( 49,50 ) . we have shown that effective end - capping of mammalian telomeres requires the non - homologous end - joining ( nhej ) protein dna - dependent protein kinase ( dna - pk ) ( 4 ) . mutation of any of the genes comprising dna - pk , i.e. ku70 , ku86 , or the catalytic subunit , dna - pkcs ( 51 ) , leads to spontaneous chromosomal end - to - end fusions that maintain large blocks of telomeric sequence at the points of fusion ( figure 1 ) . dna - pk has since been shown to associate with human telomeric dna in vivo ( 52,53 ) . events at the extreme terminus of the chromosome that normally serve to create a functionally protected telomere fail in the absence of dna - pk , resulting in inappropriate end - to - end fusion events of uncapped telomeres fusing not only to each other , but also to ir - induced dsbs ( 54 ) . we have also shown that the kinase activity of dna - pkcs is required for effective telomere protection , just as it is for nhej ( 55,56 ) . however , as with nhej , the critical in vivo substrates are not known . the in vivo consequences of inappropriate interstitial blocks of telomere sequence are also unknown , but they most likely affect chromatin stability , and in vitro studies have demonstrated they increase chromosomal instability ( 57 ) . we find that telomere fusions contribute significantly to the background level of chromosomal aberrations , and that they occur despite the presence of ample telomere sequence . thus , they are obviously not a consequence of telomere shortening , nor are they telomere associations [ defined as distinct telomere signals separated by less than approximately one - third the width of a chromatid , ( 58 ) ] . banding studies demonstrated that any chromosome could be involved in the telomere fusions ( end - capping failure is not chromosome specific ) , and further , that some of the telomere fusions were clonal , suggestive of covalent linkages ( s. bouffler , unpublished data ) . utilizing the strand - specific co - fish technique ( 59,60 ) , we found that the telomere fusions in dna - pkcs - deficient cells exclusively involved telomeres synthesized via leading - strand dna synthesis , suggesting a crucial difference in the post - replicative protection of telomeres that is linked to their mode of replication ( 61 ) . additional support for a model of end - specific differences in telomeric end - protection was provided by the demonstration of preferential loss of lagging - strand telomeres with wrn deficiency in human cell lines ( 62 ) . it protects the 3 single - stranded g - rich overhang and is involved in t - loop formation , perhaps facilitating invasion of the 3 single - stranded overhang ( 14 ) . inhibition of trf2 induces a dramatic telomere fusion phenotype ( 65 ) resulting from failure of end - protection preferentially at leading - strand telomeres ( 61 ) . end - capping failure occurs after replication , as evidenced by the presence of numerous chromatid - type telomere fusions ( 61 ) ( figure 2 ) . it has also been shown that dna damage foci form at telomeres uncapped by trf2 inhibition ( telomere dysfunction - induced foci ; tifs ) , and consistent with the cytogenetic results , uncapping of telomeres occurs in late s / g2 , i.e. after replication ( 66 ) . in addition to loss of end - capping function , chromosomal fusion can also result from the absence of sufficient telomeric repeat sequences to form a functional telomere . the most obvious mechanism for the loss of telomeric repeat sequences is attrition due to the failure to compensate for the gradual loss of telomeric repeat sequences during cell division . this failure to maintain telomere homeostasis can result from either insufficient telomerase activity or alterations in other telomere - associated proteins required for the recruitment of telomerase to the telomere . the gradual loss of telomeric repeat sequence due to insufficient telomerase activity is exemplified by the telomere shortening that occurs in telomerase - deficient somatic cells with each cell division , although due to cell senescence or apoptosis , this does not normally result in chromosome instability . in addition to gradual loss , telomeric repeat sequences can also be lost through stochastic processes , in which large blocks of telomeric repeat sequences are lost in single events . stochastic events leading to telomere loss can occur through a variety of different mechanisms , the most obvious being large deletions involving recombination , problems encountered during dna synthesis or inefficient dna repair . replication forks stall near telomeres in yeast , and require the rrm3 helicase , which promotes replication through regions of non - histone chromatin ( 67,68 ) . mammalian telomeres may also pose problems for dna replication , since the mammalian telomere - binding proteins trf1 and trf2 can inhibit replication fork movement ( 69 ) . studies in yeast have also demonstrated that telomeric regions are deficient in repair of dsbs . the introduction of dsbs at different sites along a chromosome with the i - scei endonuclease demonstrated that dsbs near telomeres are not repaired efficiently by nhej , but instead result in complex chromosome rearrangements ( 70 ) . mammalian telomeres have been shown to be deficient in repair of single - strand breaks ( 71 ) and get damaged from ultraviolet light ( 72 ) . studies with mouse es cells ( 73,74 ) and human tumor cell lines ( j. p. murnane , unpublished data ) also show that a single dsb generated by i - scei often results in complex chromosome rearrangements not commonly observed at i - scei - induced dsbs at other locations within chromosomes ( 7577 ) . cells containing mutations in various proteins known to be involved in telomere maintenance have provided valuable insights into the mechanisms of telomere loss . one such protein is wrn , which is responsible for the human genetic disease werner syndrome ( ws ) , an autosomal recessive genetic disease presenting a wide range of phenotypic abnormalities , including characteristics of premature aging ( 78 ) . wrn is a member of the recq dna helicase family and has both a 3 to 5 helicase activity and a 3 to 5 exonuclease activity ( 7981 ) . cells isolated from individuals with ws exhibit shortened life span in culture ( 82 ) , as well as an increased rate of dna rearrangements , including translocations , deletions and dicentrics ( 8388 ) . wrn binds to a number of proteins involved in recombination , including replication protein a ( rpa ) ( 89,90 ) , pcna and topoisomerase i ( 91 ) , dna polymerase delta ( 92 ) , and co - localizes with rad51 ( 93 ) and the mre11/rad50/nbs1 ( mrn ) complex in response to dsbs ( 94 ) . consistent with these protein interactions , ws cells have been shown to have a defect in homologous recombination ( hr ) ( 95,96 ) . in addition , the observations that ws cells have a prolonged s - phase ( 97 ) and wrn co - localizes with rpa in cells arrested in s - phase with hydroxyurea ( 98 ) , suggest that ws cells have a defect in resolving stalled replication forks ( 80,98,99 ) . wrn has also been demonstrated to bind the dna - pk complex ( 94,100103 ) and fen-1 ( 104 ) , proteins involved in nhej . moreover , the activity of wrn is influenced by its binding to the dna - pk complex ( 101,102 ) . thus , in addition to a role in hr , wrn also appears to have a role in the nhej pathway for dsb repair . the role of telomeres in cellular senescence initially led to the proposal that the shortened life - span of ws cells in culture might be due to accelerated telomere shortening ( 105 ) . ws cells were found to have accelerated telomere shortening , although the premature senescence in ws cells was found to occur when telomeres were longer than in senescent normal cells ( 106 ) . however , a subsequent study found that ws cells at senescence have telomeres that are similar in length to normal senescent cells ( 107 ) . combined with the fact that ws cells can be immortalized by expression of telomerase ( 107109 ) , these results suggest that ws cells have a defect in telomere maintenance that leads to premature senescence . although due to their long telomeres , mice deficient in wrn alone do not demonstrate the premature aging phenotype observed in humans with ws , later generations of telomerase - deficient mice with shortened telomeres demonstrate classic ws - like premature aging , accelerated replicative senescence , and genomic instability ( 110 ) . the mechanism responsible for telomere shortening in wrn - deficient cells has yet to be determined . one study found that cells expressing dominant - negative wrn showed an increase in chromosome ends without detectable telomeres even though no difference in average telomere length was observed ( 111 ) , indicating that telomere loss in ws is due to a stochastic process . the association of wrn with the dna - pk complex involved in nhej would suggest that telomere loss in ws cells is due to a deficiency in repair of dsbs near telomeres . however , the dna - pk complex also has a role in telomere capping ( 46,53,112,113 ) . in addition , wrn has been found to bind to trf2 ( 114 ) , which is essential for maintaining the cap on the end of the chromosome ( 65 ) , and wrn is required for d - loop resolution regulated by trf1 and trf2 ( 114 ) . based on these observations , another possible mechanism for telomere loss in wrn - deficient cells would involve t - loop deletions similar to those observed in cells deficient in trf2 ( 115 ) . however , loss of end - capping structure would not lead directly to loss of telomeric repeat sequences . a critical clue to the mechanism of telomere loss in ws cells comes from a recent study demonstrating the preferential loss of the lagging - strand of telomeres ( 62 ) , suggesting that telomere loss in ws cells is a result of the requirement for wrn in replication of the g - rich dna found in the lagging strand . this model is consistent with the involvement of wrn in dna replication and/or resolution of stalled replication forks ( 80,98,99 ) , since failure to resolve stalled replication forks can result in dna dsbs ( 116,117 ) . the importance of wrn in replication and/or recombination of telomeres is also apparent from its requirement in suppression of sister chromatid exchange ( sce ) specifically within telomeric dna ( t - sce ) ( 118 ) and activation of the alt pathway that involves recombination ( 119 ) , similar to that proposed for the yeast homolog for wrn , sgs1 ( 120122 ) . mutations in nbs1 are responsible for the autosomal recessive disease nijmegen breakage syndrome ( nbs ) , which displays a wide range of phenotypic abnormalities , including premature aging , increased cancer incidence , chromosomal instability and sensitivity to ir ( 123,124 ) . nbs1 is part of the mrn complex that also contains the mre11 and rad50 proteins ( 125,126 ) . the mrn complex is a key player in the cellular response to dsbs in that association of the mrn complex with dsbs is required for the localization and activation of atm ( 127 ) , which in turn phosphorylates nbs1 ( 128130 ) . as a result , similar to cells deficient in atm , mammalian cells deficient in nbs1 lack the s - phase cell cycle checkpoint ( 131 ) and are sensitive to ir ( 132 ) . in addition to its roles in dna recombination and repair , the mrn complex also functions in telomere maintenance . inhibition of nbs1 by rnai resulted in an increased frequency of telomere association ( 58 ) . primary fibroblasts from individuals with nbs have shortened telomeres , which are proposed to play a role in the pathology of this disease ( 133 ) . in this respect nbs is similar to at , where accelerated telomere shortening is observed in primary fibroblasts ( 134 ) , although no difference in telomere length is evident in immortal cells actively maintaining their telomeres ( 135 ) . in fact , mice with combined knockouts in both atm and the rna component of telomerase show accelerated telomere loss and premature aging , leading to the hypothesis that telomere loss is the reason for some of the phenotypic abnormalities observed in at ( 136 ) . like wrn , similar to wrn , the mrn complex interacts with trf2 ( 137 ) , and therefore is likely to function in proper end - cap formation . however , although mre11 and rad50 are found at the telomere throughout the cell cycle , nbs1 is associated with the telomere only during s - phase , suggesting that it is involved in telomere replication . thus , like wrn , a defect in nbs1 may promote telomere loss through problems in dna replication or the resolution of stalled replication forks in telomeric regions . in fact , wrn associates with mrn ( 94 ) , and both dominant - negative nbs1 ( 138 ) and wrn ( 111 ) have been found to cause similar increases in the rate of telomere loss with no change in average telomere length . selectable marker genes adjacent to telomeres have been used to study the consequences of telomere loss in mammalian cells ( 139 ) . this approach has the advantage of following the changes in individual chromosomes from the initial event , rather than attempting to reconstruct the sequence of events involved in the generation of complex rearrangements . these marked telomeres contain a herpes simplex virus thymidine kinase ( hsv - tk ) selectable - marker gene to select for loss of the telomere , as well as an 18 bp recognition site for the i - scei endonuclease to introduce dsbs , which has been widely used to study dna repair and recombination in mammalian cells ( 7577 ) . using this system , the types of chromosome rearrangements resulting from telomere loss have been followed in both mouse es cells ( 73 ) and the ej-30 human tumor cell line ( 140142 ) . in both the mouse es cells and ej-30 , telomere loss resulted in either a telomere added directly on to the end of the broken chromosome or inverted repeats resulting from sister chromatid fusion . while the addition of a new telomere resulted in stabilization of the marker chromosome , sister chromatid fusion was followed by breakage / fusion / bridge ( b / f / b ) cycles and amplification of subtelomeric dna ( figure 3 ) . these b / f / b cycles occur when the chromosome that has lost a telomere is replicated , and the sister chromatids fuse together at their ends . the fused sister chromatids then form a bridge that breaks during anaphase when the two centromeres are pulled in opposite directions . following dna replication in the next cell cycle , the sister chromatids fuse once again , and therefore these b / f / b cycles continue until the marker chromosome acquires a new telomere . because neither direct telomere addition nor sister chromatid fusion have been observed at dsbs generated by i - scei at interstitial sites ( 7577 ) , these results suggest that there is something different about the processing of dsbs occurring near telomeres . consistent with this conclusion , dsbs generated by i - scei are poorly repaired by nhej near telomeres in yeast , and result in complex chromosome rearrangements ( 70 ) . moreover , direct telomere addition on to the ends of broken chromosomes in yeast preferentially occurs near existing telomeric repeat sequences ( 143 ) . one important difference between the mouse es cells and the ej-30 human tumor cell line is that while telomeres in mouse es cells are highly stable ( loss of the hsv - tk gene < 10 events / cell / generation ) , the telomeres in the human ej-30 tumor cell line are lost at a relatively high rate ( 10 events / cell / generation ) . similar results were observed with other human tumor cell lines ( j. p. murnane , unpublished data ) . this observation is consistent with other studies demonstrating that cancer cells commonly have telomere instability ( 144,145 ) . therefore , although chromosomal rearrangements due to telomere loss in cancer cells are commonly thought to result from the extensive chromosome fusion that occurs during crisis ( 146,147 ) , a high rate of telomere loss is often observed even in human tumors and tumor cell lines that express telomerase ( 73,140,144,148,149 ) , suggesting that many tumor cells have a fundamental defect that promotes telomere loss . another important difference between the mouse es cells and human tumor cell lines is that while the direct addition of a telomere at the site of the break is a common event it mouse es cells ( 74 ) , telomere loss in human tumor cell lines often results in sister chromatid fusion followed by b / f / b cycles ( 140 ) . in addition , while b / f / b cycles last only a few generations in mouse es cells ( 73,74 ) , in ej-30 they can last for many cell generations ( 73,140,141 ) . this inability to terminate b / f / b cycles is likely to contribute to the chromosome instability resulting from telomere loss in human tumor cells . the prolonged b / f / b cycles in the ej-30 human tumor cell line results in extensive dna amplification and terminal deletions of dna on the end of the marker chromosome that lost its telomere ( 141 ) . the fused sister chromatids most often break within 1 mb of the site of fusion , which was confirmed by a subsequent study that found that anaphase bridges formed by sister chromatid fusions most often break near their center , regardless of length ( 150 ) . as a result , the region amplified by b / f / b cycles without selection is most often relatively small ( i.e. < 1 mb ) . however , some breaks also occur far from the site of fusion , resulting in large duplications and deletions ( 73,140,141 ) . this type of break is involved in the amplification of selectable marker genes located far from the end of the chromosome , and has been shown to occur at the location of fragile sites ( 151,152 ) . thus , the loss of a telomere can result in the amplification of genes anywhere on the arm of a chromosome . b / f / b cycles end when the chromosome acquires a telomere and again becomes stable . the most common mechanism for telomere acquisition in both the mouse es cells and the ej-30 tumor cell line was through translocation of the ends of other chromosomes ( 139,142 ) . in ej-30 these translocations were either non - reciprocal ( nrt ) or involved duplications , as determined by the status of the donor chromosome , i.e. with nrts , one of the homologs of the donor chromosome is missing part or all of an arm and its telomere , whereas with duplications , both homologs are intact . both types of events have important consequences for the genome as a whole . the translocations involving duplications commonly involve large portions of the arms of other chromosomes , and therefore generate allelic imbalances involving a large number of genes . on the other hand , nrts result in the loss of a telomere on the donor chromosome , resulting in its instability and the eventual acquisition of a telomere through translocations from other chromosomes . in fact , in one cell this transfer of instability was found to involve six different chromosomes , demonstrating that the loss of a single telomere can result in instability involving multiple chromosomes . therefore , in addition to amplification of dna at the end of a chromosome that has lost a telomere , once initiated , b / f / b cycles can result in other rearrangements , not only involving the chromosome that initially lost its telomere , but other chromosomes as well . in view of the fact that b / f / b cycles can continue for many cell generations , the loss of even a single telomere can generate a wide variety of chromosomal changes in the cell population . taken together , the above results demonstrate that loss of telomeric function  whether due to loss of sequence or loss of structure  and the ensuing instability and chromosomal rearrangements , can be expected to have a dramatic impact on the stability of the genome . therefore , in view of increasing evidence implicating genomic instability in carcinogenesis ( 153,154 ) , telomere loss is likely to be a significant contributing factor . a role for loss of functional telomeres in the chromosome instability commonly associated with cancer is supported by the large increase in human - like carcinomas in mice deficient in both telomerase and p53 , and the presence of chromosome rearrangements typical of b / f / b cycles in these tumors ( 146,147,155,156 ) . understanding the mechanisms of telomere maintenance and the various factors that promote telomere instability should therefore provide valuable insights into both human genetic disease and cancer . strand - specific co - fish detection of leading- ( red ) and lagging- ( green ) strand telomeres demonstrating chromosomal telomere - telomere fusion in a dna - pkcs deficient background , indicative of end - capping failure owing to loss of structure , not loss of sequence . co - fish detection of chromatid - type telomere fusions in trf2 deficient cells demonstrating preferential failure of end - capping at leading - strand ( red ) telomeres . b / f / b cycles as a mechanism for chromosome instability resulting from telomere loss . b / f / b cycles are initiated when sister chromatids fuse following the loss of a telomere . owing to the presence of two centromeres , the fused sister chromatids break when the cell attempts to divide up its sister chromatids at anaphase . because the break does not occur exactly at the site of the fusion , one daughter cell will receive a copy of the chromosome with an inverted repeat at its end , while the other daughter cell will have a copy of the chromosome with a terminal deletion . owing to the lack of a telomere , these chromosomes will again undergo sister chromatid fusion after dna replication , resulting in additional amplification and terminal deletions . the location of telomeres ( squares ) , centromeres ( circles ) and orientation of the subtelomeric sequences ( horizontal arrows ) are shown .