slx4 is a scaffold protein that binds to three dna repair endonucleases , mus81-eme1 , xpf - ercc1 , and slx1 ( andersen et  al . , 2009 ,
, 2009 , muoz et  al . , 2009 , saito et  al . , 2009 ,
the slx4 complex is required for the efficient repair of dna interstrand crosslinks ( icls ) , ( fekairi et  al . , 2009 ,
2009 ) , and the available evidence strongly suggests a role in processing dna recombination intermediates during icl repair .
the importance of slx4 for icl repair was underscored by the findings that biallelic mutations in slx4 in humans causes fanconi anemia ( fa ) ( kim et  al .
many dna repair proteins form subnuclear  foci  at sites of dna damage , but slx4 overexpressed in epitope - tagged form localizes to subnuclear foci even without dna damage ( svendsen et  al . , 2009 ) .
it was suggested that these foci correspond to telomeres , regions of repetitive dna at chromosome ends , which protect the ends from degradation ( svendsen et  al . , 2009 ) .
telomeres terminate in an overhang that is thought to invade adjacent duplex telomeric repeats to form a telomeric ( t ) loop so that the chromosome ends are not perceived as dna breaks .
an additional layer of telomere protection is afforded by a multiprotein complex called shelterin , that binds to telomeric dna ( palm and de lange , 2008 ) . in normal somatic cells , telomeres shorten with every cell division , and telomere shortening contributes to organismal aging by limiting the proliferative capacity of adult stem cells ( blasco , 2007 ) . in immortalized cells and in cancers , telomere length is maintained by telomerase , a reverse transcriptase that can add telomere repeats with the aid of an associated rna template ( greider and blackburn , 1989 , mocellin et  al . , 2013 ) .
some other immortalized cells , cancer cells , and even normal somatic cells can lengthen telomeres in a telomerase - independent manner using the alt ( alternative lengthening of telomeres ) pathway , which probably involves recombination ( bryan et  al .
first , when overexpressed in a special clone of hela cells with abnormally long telomeres slx4 forms subnuclear foci that colocalize with shelterin ( svendsen et  al . , 2009 ) .
second , slx4 was identified in a global screen for proteins that bind telomeres ( djardin and kingston , 2009 ) .
also , slx4 promotes telomere cleavage in cells lacking the rtel1 helicase ( vannier et  al . , 2012 ) . in rtel1 cells , replisomes stall in telomeres leading to cleavage of t loops and the production of extrachromosomal t circles , which is mediated by the slx4 complex ( vannier et  al . , 2012 ) .
although these observations link the slx4 complex to telomeres , it is not known whether endogenous slx4 localizes at telomeres , if this is applicable in cells that differ in the mechanisms used to maintain telomere length , or how slx4 is recruited to chromosome ends .
furthermore , in rtel1-proficient cells it remains to be determined if slx4 has a physiological role to play in telomere homeostasis . here
we first tested if endogenous slx4 localizes at telomeres . using antibodies raised in - house
, we found that endogenous slx4 forms subnuclear foci in u2os cells without addition of genotoxins ( figure  1a ) .
these foci are specific for slx4 because they disappeared when cells were transfected with slx4-specific small interfering rnas ( sirnas ) ( figure  s1a ) .
endogenous slx4 foci did not appear to be restricted to any particular cell - cycle stage , and they did not increase in number or intensity after exposure of cells to genotoxins ( data not shown ) . to investigate if endogenous slx4 foci correspond to telomeres , we tested colocalization with subunits of shelterin .
most ( around 80% ) , but not all , endogenous slx4 foci colocalized with trf2 in u2os cells ( figure  1a ) , and also with trf1 and with a peptide - nucleic acid ( pna ) probe specific for telomeric dna ( figure  1b ) . when signal intensity was measured along a straight line in a single z - section through the nucleus , the peaks corresponding to slx4 overlap with trf2 ( figure  1c ) .
it is important to note that , whereas all trf2 foci in u2os cells contained slx4 , a proportion of slx4 foci did not overlap with trf2 ( figure  1a ) .
these observations indicate that slx4 is found at all telomeres in u2os cells , but a proportion of the slx4 foci do not correspond to telomeres .
slx4 associates with three separate structure - specific nucleases , xpf - ercc1 , mus81-eme1 , and slx1 .
xpf - ercc1 was previously shown to localize at telomeres ( zhu et  al . , 2003 ) , and in this light we found that all of the xpf - ercc1 foci in cells colocalize with slx4 ( figures s1b and s1d ) .
mus81-eme1 was also shown to localize at telomeres ( zeng et  al . , 2009 ) , but only around 5% of u2os cells show mus81 foci colocalizing with slx4 ( figures s1c and s1d ) .
endogenous slx1 foci were only barely detectable with our anti - slx1 antibodies ( data not shown ) .
slx4 controls the stability of slx1 ( muoz et  al . , 2009 ) , and
overexpression of a gfp - tagged form of slx4 increased the levels of endogenous slx1 ( data not shown ) . under these conditions ,
endogenous slx1 foci could be detected with anti - slx1 antibodies ( figure  1d ) , and most of these foci colocalize with trf2 ( data not shown ) . in contrast , slx1 foci could not be detected when we overexpressed a mutant form of slx4 ( c1805r ) that is incapable of interacting with slx1 ( figure  1d ) ( d.c .
we also found that a gfp - tagged form of slx1 formed foci that colocalized with trf2 ( figure  1e ) .
taken together , these data show that the endogenous slx4 complex localizes at telomeres . to confirm the association of slx4 with telomeres
telomeric dna , but not alu repeat dna , was detected in slx4 immunoprecipitates of u2os cells using two separate anti - slx4 antibodies raised in sheep , but not when nonspecific sheep immunoglobulin ( ig ) g or anti - cenpa antibodies were used .
the chip signal in slx4 precipitates was comparable to the signal in trf2 precipitates ( figure  2a ) .
we also detected telomeric dna , but not alu dna , in anti - slx1 immunoprecipitates in u2os cells ( figure  2b ) .
telomere length is maintained by the alt pathway in u2os cells , whereas in other cell lines telomere length can be maintained by telomerase ( cesare and reddel , 2010 ) .
chip experiments showed that slx4 associates with telomeric dna , but not alu dna , in a549 and hela epithelial cells , two non - alt cell lines that are telomerase positive ( figures 2c and 2d , left panels ) .
the chip signal in anti - slx4 immunoprecipitates from these cells was weaker than in u2os cells , probably because a549 and hela cell telomeres are shorter than in u2os cells ( lee et  al . , 2008 ) .
nonetheless , the chip signal in slx4 precipitates from a549 and hela cells was comparable to the signal in trf2 precipitates ( figures 2c and 2d , left panels ) .
furthermore , slx4 foci in a549 cells and hela cells are considerably less intense than in u2os cells , but a significant proportion of the foci still colocalized with trf2 ( figures 2c and 2d , middle panels ) .
when signal intensity along an arbitrary track in a single z - section of the nucleus was quantitated , the peaks corresponding to slx4 and trf2 in a549 and hela cells demonstrated good overlap ( figures 2c and 2d , right panels ) .
we also observed slx4 foci in ccl-211 primary lung fibroblasts , and some of these colocalize with trf1 similar to hela cells ( figure s1e ) .
although a significant proportion of telomeric foci in these cells do not contain slx4 , when signal intensity along an arbitrary track in a single z - section of the nucleus was quantitated , the peaks corresponding to slx4 demonstrated reasonably good overlap with trf1 ( figure  s1e ) .
taken together , these data show that the slx4 complex localizes at telomeres in a range of human cell lines that differ in the mechanisms used to maintain telomere length .
both subunits of the trf2-rap1 shelterin subcomplex were found previously in slx4 immunoprecipitates ( muoz et  al . , 2009 , svendsen et  al . , 2009 ) .
we next sought to determine whether slx4 is recruited to telomeres by either of these two proteins .
yeast two - hybrid analysis , using xpf as a positive control , revealed that slx4 binds to trf2 , not rap1 ( figure  3a ) , and it seemed likely therefore that slx4 is recruited to telomeres by trf2 .
to investigate this possibility , we sought mutations in slx4 that abolish the interaction with trf2 , and our search was aided by a previous report describing a motif found in a range of proteins that interact with trf2 ( f / y - x - l - x - p ; figure  3b ) ( chen et  al . , 2008 ) .
inspection of the primary amino acid sequence of human slx4 failed to identify a classical trf2-binding motif ( tbm ) , but we found a tbm - like motif ( h - x - l - x - p ) in which the first residue ( f / y ) is replaced by a histidine residue , which like f and y contains a planar aromatic ring structure ( figure  3b ) .
this motif in slx4 lies after the btb domain in an unstructured region of the protein ( figure  3b , data not shown ) .
we next tested the effect of mutating the key residues in the putative tbm in human slx4 on its interaction with trf2 .
to this end , u2os cells were cotransfected with rfp - tagged trf2 and ha - tagged slx4 wild - type or ha - slx4 in which h1020 , l1022 , or p1024 were mutated to alanine .
whereas wild - type ha - slx4 coprecipitated with rfp - trf2 , mutation of h1020 , l1022 , or p1024 caused a major reduction in the amount of ha - slx4 coprecipitating with rfp - trf2 ( figure  3c ) .
similar results were obtained when gfp - tagged slx4 was immunoprecipitated from cells coexpressing rfp - trf2 ( figure  3d ) .
importantly , mutating h1020 , l1022 , or p1024 had no effect on the ability of slx4 to interact with xpf - ercc1 , mus81 , or slx1 ( figure  s2a )
. taken together , these data show that a tbm in human slx4 mediates interaction with trf2 .
intriguingly , a clear tbm is found in slx4 in primates but not in mammals lower down the evolutionary tree ( figure  s2b ) .
slx4 orthologs in some mammals have a motif that is vaguely similar to the tbm in primates ( figure  s2b ) , but none of the mammalian motifs would be expected to interact with trf2 based on previous work ( chen et  al . , 2008 ) .
we next tested if mutating the tbm in slx4 affects its localization at telomeres . to this end ,
gfp - tagged wild - type slx4 , or gfp - slx4-l1022a , was stably expressed in u2os cells and foci were analyzed .
surprisingly , the overall number of foci formed by the slx4 l1022a mutant was similar to wild - type slx4 ( figures 3e and 3f ) .
however , whereas almost 65% of wild - type gfp - tagged slx4 foci colocalized with trf2 , only around 10% of foci formed by the slx4 l1022a mutant coincided with trf2 , and more than 40% of the foci clearly did not ( figures 3e and 3f ) .
the remaining 40% of slx4l1022a foci did not colocalize with trf2 , but were found adjacent to , but distinct from trf2 foci ( figures 3e and 3f ) . to further test the impact of mutating the slx4 tbm on telomere binding , we used chip analysis , which revealed that mutating slx4 l1022 to alanine caused an almost 80% reduction in the amount of telomeric dna associated with slx4 ( figures 3 g and 3h ) .
we tested this idea further using a dominant - negative form of trf2 ( trf2bm ) that heterodimerizes with endogenous trf2 blocking its binding to dna ( van steensel et  al . , 1998 ) .
as shown in figure  s2c , overexpression of an  rfp - tagged form of trf2bm , but not wild - type trf2 , caused a substantial reduction in the number of slx4 foci in u2os cells ( figure  s2c ) .
some of the cells transfected with trf2bm did not express this protein , and slx4 foci were normal in these cells ( figure  s2c ) .
taken together , these data indicate that trf2 recruits slx4 to telomeres in human cells .
we also tested if the slx4-trf2 interaction is required for association of slx1 with telomeres .
we showed earlier that endogenous slx1 forms trf2-coincident foci in cells overexpressing wild - type slx4 ( figure  1d ) .
slx1 also forms foci in cells overexpressing slx4-l1022a , but , although these foci colocalize with slx4-l1022a , they do not colocalize with trf2 ( figure  s3a ) .
slx4 is required for efficient dna repair . because trf2 has been shown to localize at sites of dna damage ( huda et  al . ,
2012 ) , and to promote dna repair ( huda et  al . , 2009 ) , we tested the possibility that trf2 might recruit slx4 to dna damage sites as well as to telomeres . to this end , we induced dna damage using local laser microirradiation and then checked by confocal microscopy for the ability of slx4 to form  laser stripes . 
time - lapse experiments revealed that gfp - tagged slx4 formed subnuclear stripes along the track of dna damage induced by laser irradiation visible within 12  min of irradiation , reaching a maximum intensity after 1  hr ( figure  s3b ) .
the slx4-l1022a mutant localized at tracks of laser - induced dna damage with similar kinetics and intensity to wild - type slx4 ( figure  s3b ) . we conclude from these data that trf2
we also tested if the association of slx4 with trf2 is required for dna repair using genotoxin hypersensitivity as readout . to this end , we stably expressed untagged forms of slx4 and slx4-l1022a in cells from fanconi anemia patient eufa-1354 that express abnormally low levels of an n - terminally truncated form of slx4 ( stoepker et  al .
wild - type slx4 and slx4-l1022a are indistinguishable in their ability to rescue the mitomycin - c hypersensitivity of eufa-1354 cells . taken together , the data above indicate that neither the localization of slx4 at dna damage sites , nor the ability of slx4 to promote dna repair , requires binding to trf2 .
because the l1022a slx4 mutant interacts with xpf - ercc1 , mus81-eme1 , and slx1 ( figure  s2a ) but does not localize at telomeres ( figures 3e3h ) , we reasoned that it might function as a dominant negative by sequestering the associated nucleases away from telomeres .
in this light , we made u2os cells that stably express gfp - tagged slx4 , or gfp - slx4-l1022a in a tetracycline - inducible manner .
upon induction of slx4-l1022a , we noticed an increase in the proportion of cells with  tifs  ( telomere dysfunction - induced foci ) ( takai et  al . ,
2003 ) , defined as 53bp1 foci that colocalize with trf1 , compared with cells expressing wild - type slx4 ( figure  4a ) . in contrast , there was little difference between wild - type slx4 and the slx4-l1022a mutant in terms of the induction of 53bp1 foci that did not colocalize with trf1 ( figure  4a ) .
consistent with these data , sirna - mediated depletion of slx4 from u2os cells increased the proportion of cells with tifs ( figures s4a and s4b ) .
slx4-specific sirnas 3 and 4 resulted in more efficient depletion of slx4 than sirnas 1 and 2 , and consequently they were more potent at inducing tifs .
intriguingly , the lack of an obvious trf2 binding motif in nonprimate mammalian orthologs of slx4 suggests that slx4 should not localize at telomeres in mouse cells , for example , and we next investigated this possibility .
as shown in figure  s4c , whereas human slx4 forms foci that colocalize with trf2 when ectopically expressed in mouse embryonic fibroblasts ( mefs ) , mouse slx4 forms foci but they do not colocalize with trf2 .
this is reminiscent of the human slx4-l1022a mutant that forms foci that do not colocalize with telomeres ( figure  3e3h ) .
these observations indicate that slx4 does not associate stably with telomeres in mouse cells , at least in fibroblasts .
nonetheless , we reasoned that slx4 might have roles at telomeres that do not require stable association with chromosome ends . with this in mind , we analyzed telomeres in slx4 mice generated in this laboratory , which will be described in detail elsewhere ( d.c .
first , we observed increased incidence of tifs in mouse embryonic fibroblasts ( mefs ) from slx4 mice ( figure  s4d ) .
second , telomeres in livers from slx4 mice were longer than in slx4 mice or wild - type mice ( figure  4b ) .
telomeres were also longer in mefs from slx4 mice , but lengthening was reversed by expression of mouse slx4 in these cells so that telomeres were similar in length to wild - type mefs ( figure  4c ) .
these data suggest that slx4 might play a role in trimming telomeres to prevent overlengthening .
we also found that telomeres from slx4 mefs show increased fragility assessed by the frequency of multitelomeric signals ( mtss ) compared with slx4 mefs and wild - type cells ( figure  4d ) .
the data above indicate that targeting of slx4 to telomeres prevents dna damage in human cells , and that slx4 also plays a role in telomere homeostasis in mice in a manner that does not appear to require the stable retention of the slx4 complex at telomeres .
in this study , we showed slx4 is recruited to telomeres in human cells by trf2 .
we identified a motif in human slx4 required for direct interaction with trf2 that is similar but not identical to the trf2-binding motif ( tbm ; f / y - x - l - x - p ) identified in proteins such as apollo ( chen et  al . , 2008 ) .
in the slx4 motif , however , the f / y residue is replaced by a histidine residue .
this observation extends the consensus trf2-binding motif and raises the possibility that other proteins might interact with trf2 through tbms similar to that of slx4 .
although slx4 is a rapidly evolving protein , it is somewhat surprising that the slx4 tbm - like motif is conserved in primates but not in mammals lower down the evolutionary tree ( figure  s2b ) .
the absence of a tbm , however , does not exclude the possibility that slx4 ( or an slx4-associated protein ) in nonprimate species associates with trf2 and/or telomeres by alternative mechanisms .
however , we have been unable to detect endogenous slx4 at telomeres in mouse embryonic fibroblasts , and , whereas human slx4 overexpressed in mefs forms telomeric foci , mouse slx4 does not ( figure  s4c ) .
therefore , slx4 associates with trf2 and telomeres in primates but not in other mammals . in human cells , preventing localization of slx4 at telomeres caused signs of dna damage ( figure  4a ) . the precise nature of this damage is not yet clear , but it might result from difficulties in one of the many processing steps during and after dna replication necessary for establishing the correct structure at telomeres .
it appears that not all of the telomere - related functions of the slx4 complex require stable association with telomeres , because telomeres are longer and more fragile in organs and cells from slx4 mice and telomere lengthening in slx4 mefs can be reversed by expression of wild - type slx4 ( figures 4b and 4c ) . from this perspective
telomere trimming ,  a mechanism that has been proposed to prevent overlengthening of telomeres ( pickett and reddel , 2012 ) .
it is not yet clear which of the slx4-associated nucleases are required to prevent telomere lengthening .
it is unlikely that xpf - ercc1 is the relevant nuclease because telomere length is not affected in ercc1 mice ( zhu et  al . , 2003 ) .
the nuclease that promotes slx4-dependent telomere shortening could be mus81-eme1 and/or slx1 ; it will be interesting to test the relevant knockout mice for abnormally long telomeres .
slx1 is a good candidate considering the boulton lab has shown that it is required for the production of t circles in cells lacking rtel1 ( vannier et  al . , 2012 ) .
the consequences of overlong telomeres for cell function and organism function are not clear , but this will be interesting to study .
u2os cells expressing gfp - slx4 were preextracted in csk buffer : 10  mm pipes ( ph 6.8 ) containing 100  mm nacl , 300  mm sucrose , 3  mm magnesium chloride , 1  mm egta , and 0.5% [ v / v ] triton x-100 , fixed with 2% paraformaldehyde ( pfa ) ( ph 7.0 ) , permeabilized with pbs containing 0.5% ( v / v ) triton x-100 , blocked with bsa ( 3% w / v ) in pbs , and probed with primary antibodies for 1  hr at room temperature . after extensive washing
, cells were incubated with secondary alexa antibodies for 2  hr at room temperature .
wide - field image stacks of cells were acquired using a deltavision microscope ( applied precision ) and subjected to iterative deconvolution to remove out - of - focus light and were further processed in omero ( allan et  al . , 2012 ) . for endogenous slx4 immunofluorescence , cells grown on poly - l - lysine - coated coverslips were preextracted in csk buffer or pbs containing 0.5% triton x-100 prior to fixation in 3% pfa in csk ( ph 7.0 ) .
cells were permeabilized in pbs - containing 0.5% ( v / v ) triton x-100 and then blocked in antibody dilution buffer ( abdil ) : pbs containing 5% normal donkey serum , 0.1% fish skin gelatin , 0.1% triton x-100 , 0.05% tween-20 , and 0.05% sodium azide , before incubation with primary antibodies in abdil for 1  hr at room temperature or overnight at 4c .
after washing , cells were incubated with the relevant secondary antibodies for 2  hr at room temperature , stained with dapi , and mounted on glass slides .
cells grown to subconfluency on 15  cm plates were crosslinked with 1% ( v / v ) formaldehyde for 15  min , with 0.125  m glycine for 5  min , and then washed twice with pbs .
cells ( 4   10 cells per ip ) were lysed in chromatin immunoprecipitation ( chip ) lysis buffer ( 1% sds , 10  mm edta , 50  mm tris - hcl [ ph  8.0 ] plus protease inhibitors ) , followed by sonication for 15  s at 50% amplitude and 30  s rest ; this was repeated eight times at 4c before cells were centrifuged at 12,000  rpm at 4c for 10  min .
cell supernatants were diluted 1:10 in chip dilution buffer ( 1% [ v / v ] triton x-100 , 2  mm edta , 150  mm nacl , and 20  mm tris - hcl [ ph 8.0 ] ) and precleared with 2  g of sheared salmon sperm dna ( invitrogen ) and 20  l protein g - sepharose beads per equivalent ip volume ( 2  ml ) .
the supernatant was incubated with the relevant antibodies overnight , before the addition of protein g - sepharose beads for 1  hr .
after extensive washes , immune complexes were eluted with elution buffer ( 100  mm nahco3 and 1% sds ) and incubated overnight at 65c to reverse crosslinks .
protein and rna were removed by incubation with proteinase k and rnase a at 37c for 1  hr .
dna was purified by extraction with phenol / chloroform / isoamyl alcohol ( 25:24:1 ) , followed by centrifugation and retrieval of the aqueous supernatant .
dna was precipitated with ice - cold ethanol ( 96% v / v ) , washed with 70% ethanol , and resuspended in 100  l of water .
extended experimental procedurescell culture , fractionation , and lysisu2os cells were cultured in dmem growth medium ( gibco ) supplemented with 10% ( v / v ) fetal bovine serum , 100 units / ml penicillin , 100  g / ml streptomycin and 1% l - glutamate ( gibco , invitrogen ) .
u2os cells stably expressing gfp - slx4 were generated using the flp - in t - rex system ( invitrogen ) as described previously ( muoz et  al . , 2009 ) .
cells were maintained in selective media containing 100  g / ml hygromycin and 15  g / ml blasticidin .
protein expression was induced by adding tetracycline to 0.1  g / ml for 24  hr . for immunoprecipitations and western blotting ,
cells were lysed in 50  mm tris - hcl ph 7.4 containing 150  mm nacl , 1  mm edta ph  8 , 1  mm egta ph 8 , 1% ( v / v ) triton x-100 , 270  mm sucrose supplemented with -mercaptoethanol ( 0.1%v / v ) , benzonase ( 50mu/l ) and 1 roche protease inhibitor tablet per 50  ml of buffer.slx4 micethe generation of slx4 mice will be described elsewhere ( d.c . and j.r , unpublished data ) .
animals were housed under specific pathogen free conditions in accordance with uk and eu regulations .
all procedures were carried out in accordance with university of dundee and united kingdom home office regulations.isolation and immortalization of mefsday 13.5 embryos were derived from timed matings between heterozygous parents . after decapitation ,
the red organs were removed , the embryo was minced and resuspended in 1  ml trypsin and incubated at 37c for 15  min before the addition of 10  ml growth medium .
cells were plated and allowed to attach over night before cells were washed with fresh medium to remove debris .
when cells reached confluency they were split and re - plated and this was considered passage 1 .
mefs were 3t3-immortalized.sirnacells were transfected with the relevant sirna duplex ( 30  nm ) using the calcium phosphate precipitation method or hiperfect ( qiagen ) according to the manufacturer s protocol .
the slx4 mrna target sequences used in figures  s4a and s4b were : slx4 - 1 gagaagaaccctaatgaaa dtdtslx4 - 2 gcacaagggcccagaacaa dtdtslx4 - 3 ggagaaggaagcagagaat dtdtslx4 - 4 aaacgtgaatgaagcagaa dtdtslx4 - 4 was used in figure  s1a .
the final concentration of sirna added to cells was 20  nm.antibodiesthe following primary antibodies were used in this study.western blottingmouse anti - human xpf diluted 1:5000 ( thermoscientific ms1381 ) ; rabbit anti - trf2 diluted 1:1000 ( cst # 2645 ) .
sheep polyclonal slx4 antibodies were raised against a bacterially  expressed protein fragment corresponding to anti - human slx4 ( amino acids 1535 - 1834 ) fused to gst .
the antibodies were purified from sheep serum by affinity chromatography on ch - sepharose to which the peptide immunogen had been covalently coupled .
sheep polyclonal slx1 antibodies were raised against bacterially  expressed human slx1 ( full - length ) fused to gst .
the antibodies were purified from sheep serum by affinity chromatography on ch - sepharose to which the peptide immunogen had been covalently coupled.immunoprecipitationsheep anti - human slx4 ( 1535 - 1834 ) as above ; sheep anti - gfp ( as sheep igg control , raised in the dstt ) ; mouse anti - trf2 ( millipore 41794 ) ; rabbit anti - rap1 ( bethyl a300 - 306a ) ; normal mouse igg ( cst # 5415 ) ; normal rabbit igg ( cst # 2729 ) .
all antibodies were used at 4  g antibody per 2  mg of cell lysate.immunofluorescencemouse anti - trf1 diluted 1:200 ( abcam ab10579 ) ; rabbit anti-53bp1 diluted 1:500 ( a300 - 272a ) ; mouse anti - human xpf diluted 1:500 ( thermoscientific ms1381 ) ; mouse anti - trf2 diluted 1:400 ( millipore 4a794 ) ; anti - slx1 diluted 1:100 .
all secondary antibodies were from invitrogen : donkey anti - mouse alexa 594 ( diluted 1:800 ) ; donkey anti - sheep alexa 594 ( diluted 1:800 ) ; donkey anti - mouse alexa 647 ( diluted 1:500 ) ; donkey anti - rabbit alexa 647 diluted 1:500 .
sheep polyclonal slx4 antibodies for immunofluorescence were raised against a bacterially  expressed protein fragment corresponding to anti - human slx4 ( amino acids 381 - 680 ) fused to gst .
the antibodies were purified from sheep serum by affinity chromatography on ch - sepharose to which the peptide immunogen had been covalently - coupled .
these antibodies were diluted 1:200.chipsheep anti - human slx4 ( 381 - 680 ) as above ; sheep anti - human slx4 ( 1535 - 1834 ) as above ; mouse anti - trf2 ( imgenex img124a ) ; mouse anti - cenpa ( abcam ab13939 ) ; sheep anti - gfp ; rabbit anti - gfp ( abcam ab290 ) ; normal mouse igg ( cst # 5415 ) ; normal rabbit igg ( cst # 2729 ) .
all antibodies used at 3  g per chip.dot blotting and hybridizationchip dna was blotted onto hybond h membrane ( ge healthcare ) and cross - linked with 1200 j / m of uv - c light .
membranes were pre - hybridized by incubation in church buffer ( 0.5 m sodium phosphate buffer ph7.2 , 1 mm edta , 1% bsa ) for 1  hr .
membranes were then incubated with 800bp ( ttagggn ) telomere repeat probe radio - labeled with [ p]-dctp using klenow fragment , boiled before incubation in church buffer .
alternatively , membranes were incubated with a poly - nucleotide kinase ( pnk ) gamma 32p end labeled alu repeat probe ( cggagtctcgctctgtcgcccaggctggagtgcagtggcgcga ) in church buffer .
membranes were washed in 2 x ssc ( 300  mm nacl , 15  mm sodium citrate ) four times for 5  min each at 65c , and exposed to photographic film overnight at 80c.coupled immunofluorescence - fishin coupled immunofluorescence ( if)-fish experiments , immunofluorescence was carried out as normal with cells grown on coverslips .
cells were then washed twice in pbs and fixed again with 3% formaldehyde ( 5  min ) before further washing .
cells were then serially dehydrated in 70% , 95% and then 100% ethanol before drying in air for 2  min .
cells were incubated with fitc labeled telomeric fish probe ( dako ) and dishes were gently covered with saranwrap to aid distribution of the probe .
after 10  min at 80c , the coverslips were transferred to 4c and incubated overnight .
after two 15  min washes in buffer ( 10 mm tris - hcl , ph7.4 ) containing 70% ( v / v ) formamide at 50c , coverslips were washed three times for 5  min each with pbs ; the second pbs wash included dapi at 1  g / ml .
cells were then mounted for microscopic analysis.telomere fluorescence analyses of liver sectionsparaffin - embedded liver sections were hybridized with a pna - telomeric probe , and fluorescence intensity of telomeres was determined as previously described ( zijlmans et  al . , 1997 ) .
quantitative image analysis was performed on images obtained using a confocal microscope ( leica tcs - sp5 dobs ) and the definiens developer cell software ( version xd 1.2 ; definiens ag).telomere length and cytogenetic analysis using telomere q - fish on metaphase spreadsq - fish on metaphases from primary mef was performed as described previously ( herrera et  al . , 1999 ) . where indicated , mefs were treated with aphidicolin at 0.5  m for 18  hr followed by 6  hr recovery .
images were captured at 100x magnification using a cohu ccd camera on a leica dmra ( leica , heidelberg , germany ) microscope .
telomere fluorescence signals were quantified by using the tfl - telo program ( gift from peter lansdorp , vancouver , canada ) ( zijlmans et  al .
, 1997).laser microirradiationcells were plated on glass - bottomed dishes and pre - sensitized with 10  mm 5-bromo-2-deoxyuridine ( brdu , sigma - aldrich ) for 24  h at 37c .
dna damaged was induced using a 405  nm cw 30 mw diode laser scanned 50x at 100% power over the selected roi ( region of interest ) in one optical section corresponding to the middle of the nucleus .
live cell imaging was carried out on a zeiss 710 confocal microscope equipped with a 37c environmental chamber and supplied with humidified 5% co2 .
cells were imaged with a zeiss plan - apochromat 63x 1.4na oil immersion objective lens .
u2os cells were cultured in dmem growth medium ( gibco ) supplemented with 10% ( v / v ) fetal bovine serum , 100 units / ml penicillin , 100  g / ml streptomycin and 1% l - glutamate ( gibco , invitrogen ) .
u2os cells stably expressing gfp - slx4 were generated using the flp - in t - rex system ( invitrogen ) as described previously ( muoz et  al .
cells were maintained in selective media containing 100  g / ml hygromycin and 15  g / ml blasticidin .
protein expression was induced by adding tetracycline to 0.1  g / ml for 24  hr . for immunoprecipitations and western blotting
, cells were lysed in 50  mm tris - hcl ph 7.4 containing 150  mm nacl , 1  mm edta ph  8 , 1  mm egta ph 8 , 1% ( v / v ) triton x-100 , 270  mm sucrose supplemented with -mercaptoethanol ( 0.1%v / v ) , benzonase ( 50mu/l ) and 1 roche protease inhibitor tablet per 50  ml of buffer . the generation of slx4 mice will be described elsewhere ( d.c . and
animals were housed under specific pathogen free conditions in accordance with uk and eu regulations .
all procedures were carried out in accordance with university of dundee and united kingdom home office regulations .
the red organs were removed , the embryo was minced and resuspended in 1  ml trypsin and incubated at 37c for 15  min before the addition of 10  ml growth medium .
cells were plated and allowed to attach over night before cells were washed with fresh medium to remove debris .
when cells reached confluency they were split and re - plated and this was considered passage 1 .
cells were transfected with the relevant sirna duplex ( 30  nm ) using the calcium phosphate precipitation method or hiperfect ( qiagen ) according to the manufacturer s protocol .
the slx4 mrna target sequences used in figures  s4a and s4b were : slx4 - 1 gagaagaaccctaatgaaa dtdtslx4 - 2 gcacaagggcccagaacaa dtdtslx4 - 3 ggagaaggaagcagagaat dtdtslx4 - 4 aaacgtgaatgaagcagaa dtdtslx4 - 4 was used in figure  s1a .
slx4 - 1 gagaagaaccctaatgaaa dtdt slx4 - 2 gcacaagggcccagaacaa dtdt slx4 - 3 ggagaaggaagcagagaat dtdt slx4 - 4 aaacgtgaatgaagcagaa dtdt slx4 - 4 was used in figure  s1a .
the final concentration of sirna added to cells was 20  nm . the following primary antibodies were used in this study .
mouse anti - human xpf diluted 1:5000 ( thermoscientific ms1381 ) ; rabbit anti - trf2 diluted 1:1000 ( cst # 2645 ) .
sheep polyclonal slx4 antibodies were raised against a bacterially  expressed protein fragment corresponding to anti - human slx4 ( amino acids 1535 - 1834 ) fused to gst .
the antibodies were purified from sheep serum by affinity chromatography on ch - sepharose to which the peptide immunogen had been covalently coupled .
sheep polyclonal slx1 antibodies were raised against bacterially  expressed human slx1 ( full - length ) fused to gst .
the antibodies were purified from sheep serum by affinity chromatography on ch - sepharose to which the peptide immunogen had been covalently coupled .
sheep anti - human slx4 ( 1535 - 1834 ) as above ; sheep anti - gfp ( as sheep igg control , raised in the dstt ) ; mouse anti - trf2 ( millipore 41794 ) ; rabbit anti - rap1 ( bethyl a300 - 306a ) ; normal mouse igg ( cst # 5415 ) ; normal rabbit igg ( cst # 2729 ) .
all antibodies were used at 4  g antibody per 2  mg of cell lysate .
mouse anti - trf1 diluted 1:200 ( abcam ab10579 ) ; rabbit anti-53bp1 diluted 1:500 ( a300 - 272a ) ; mouse anti - human xpf diluted 1:500 ( thermoscientific ms1381 ) ; mouse anti - trf2 diluted 1:400 ( millipore 4a794 ) ; anti - slx1 diluted 1:100 .
all secondary antibodies were from invitrogen : donkey anti - mouse alexa 594 ( diluted 1:800 ) ; donkey anti - sheep alexa 594 ( diluted 1:800 ) ; donkey anti - mouse alexa 647 ( diluted 1:500 ) ; donkey anti - rabbit alexa 647 diluted 1:500 . sheep polyclonal slx4 antibodies for immunofluorescence were raised against a bacterially  expressed protein fragment corresponding to anti - human slx4 ( amino acids 381 - 680 ) fused to gst .
the antibodies were purified from sheep serum by affinity chromatography on ch - sepharose to which the peptide immunogen had been covalently - coupled .
sheep anti - human slx4 ( 381 - 680 ) as above ; sheep anti - human slx4 ( 1535 - 1834 ) as above ; mouse anti - trf2 ( imgenex img124a ) ; mouse anti - cenpa ( abcam ab13939 ) ; sheep anti - gfp ; rabbit anti - gfp ( abcam ab290 ) ; normal mouse igg ( cst # 5415 ) ; normal rabbit igg ( cst # 2729 ) .
chip dna was blotted onto hybond h membrane ( ge healthcare ) and cross - linked with 1200 j / m of uv - c light .
membranes were pre - hybridized by incubation in church buffer ( 0.5 m sodium phosphate buffer ph7.2 , 1 mm edta , 1% bsa ) for 1  hr .
membranes were then incubated with 800bp ( ttagggn ) telomere repeat probe radio - labeled with [ p]-dctp using klenow fragment , boiled before incubation in church buffer .
alternatively , membranes were incubated with a poly - nucleotide kinase ( pnk ) gamma 32p end labeled alu repeat probe ( cggagtctcgctctgtcgcccaggctggagtgcagtggcgcga ) in church buffer .
membranes were washed in 2 x ssc ( 300  mm nacl , 15  mm sodium citrate ) four times for 5  min each at 65c , and exposed to photographic film overnight at 80c . in coupled immunofluorescence ( if)-fish experiments ,
cells were then washed twice in pbs and fixed again with 3% formaldehyde ( 5  min ) before further washing .
cells were then serially dehydrated in 70% , 95% and then 100% ethanol before drying in air for 2  min .
cells were incubated with fitc labeled telomeric fish probe ( dako ) and dishes were gently covered with saranwrap to aid distribution of the probe .
after 10  min at 80c , the coverslips were transferred to 4c and incubated overnight .
after two 15  min washes in buffer ( 10 mm tris - hcl , ph7.4 ) containing 70% ( v / v ) formamide at 50c , coverslips were washed three times for 5  min each with pbs ; the second pbs wash included dapi at 1  g / ml .
paraffin - embedded liver sections were hybridized with a pna - telomeric probe , and fluorescence intensity of telomeres was determined as previously described ( zijlmans et  al . , 1997 ) .
quantitative image analysis was performed on images obtained using a confocal microscope ( leica tcs - sp5 dobs ) and the definiens developer cell software ( version xd 1.2 ; definiens ag ) .
q - fish on metaphases from primary mef was performed as described previously ( herrera et  al .
, 1999 ) . where indicated , mefs were treated with aphidicolin at 0.5  m for 18  hr followed by 6  hr recovery .
images were captured at 100x magnification using a cohu ccd camera on a leica dmra ( leica , heidelberg , germany ) microscope .
telomere fluorescence signals were quantified by using the tfl - telo program ( gift from peter lansdorp , vancouver , canada ) ( zijlmans et  al . , 1997 ) .
cells were plated on glass - bottomed dishes and pre - sensitized with 10  mm 5-bromo-2-deoxyuridine ( brdu , sigma - aldrich ) for 24  h at 37c .
dna damaged was induced using a 405  nm cw 30 mw diode laser scanned 50x at 100% power over the selected roi ( region of interest ) in one optical section corresponding to the middle of the nucleus .
live cell imaging was carried out on a zeiss 710 confocal microscope equipped with a 37c environmental chamber and supplied with humidified 5% co2 .
cells were imaged with a zeiss plan - apochromat 63x 1.4na oil immersion objective lens .
 summaryslx4 , a scaffold for structure - specific dna repair nucleases , is important for several types of dna repair . 
 many repair proteins bind to sites of dna damage , resulting in subnuclear  
 foci ,  
 but slx4 forms foci in human cells even without dna damage . using several approaches , 
 we show that most , but not all , slx4 foci localize to telomeres in a range of human cell lines irrespective of the mechanisms used to maintain telomere length . 
 the slx1 holliday - junction - processing enzyme is recruited to telomeres  by slx4 , and slx4 , in turn , is recruited by a motif that binds to the shelterin subunit trf2 directly . 
 we also show that trf2-dependent recruitment of slx4 prevents telomere damage . 
 furthermore , slx4 prevents telomere lengthening and fragility in a manner that appears to be independent of telomere association . 
 these findings reveal that slx4 plays multiple roles in regulating telomere homeostasis . 
Introduction
Results
Discussion
Experimental Procedures