this complex includes three structure - selective nucleases : xpf - ercc1 , mus81-eme1 , and slx1 ( andersen et  al . , 2009 ; fekairi et  al . , 2009 ; muoz et  al . , 2009 ; saito et  al . , 2009 ; svendsen et  al . , together these nucleases confer the complex with the ability to cleave a wide range of branched dna structures in  vitro , which mimic dna intermediates that occur during the repair of damaged dna and broken dna replication forks . both mus81-eme1 and xpf - ercc1 are required for the repair of dna interstrand crosslinks ( icls ) in mammalian cells , and the latter is also required for repair of uv - induced lesions ( ciccia et  al . , 2008 ) . depletion of slx4 from human cells using sirna duplexes does not affect uv repair but causes pronounced hypersensitivity to agents that induce dna icls ( fekairi et  al . , 2009 ; muoz et  al . , 2009 ; svendsen et  al . , the importance of slx4 in icl repair in humans is underscored by the observation that biallelic mutations in slx4/fancp cause fanconi anemia ( fa ) ( kim et  al . 2011 ) , a cancer predisposition syndrome accompanied by developmental , skeletal , and hematological defects ( auerbach , 2009 ) . despite the clear importance of the slx4 complex in dna repair , for example , it is not yet known if slx1 is involved in icl repair , partly because depletion of slx1 from human cells destabilized slx4 , preventing the functional analysis of slx1 ( muoz et  al . , furthermore , it is not yet known how slx4 affects the associated nucleases , and there has been some debate about whether the exquisite hypersensitivity of slx4-defective cells reflects the loss of regulation of one or more of these nucleases . in this light , a recent study concluded that the role of slx4 in icl repair involves xpf - ercc1 only , because a fragment of slx4 lacking amino acids 1499 , that did not interact with xpf - ercc1 , did not rescue the mitomycin - c ( mmc ) sensitivity of slx4 hypomorphic mefs ( crossan  et  al . , however , the first 499 amino acids of slx4 also contain two ubiquitin - binding domains that are vital for icl repair but that are not required for slx4 to interact with xpf ( kim et  al . two studies used slx4 deletion mutants lacking the c - terminal helix - turn - helix ( hth ) domain to investigate the importance of slx1 binding for icl repair . one study found that the hth deletion mutant fully rescues the sensitivity of slx4 hypomorphic mefs ( crossan et  al . , 2011 ) , whereas the other showed that this mutant only partly rescued the mmc sensitivity of slx4-defective fa cells ( kim et  al . , 2013 ) . therefore , the functional relevance of the binding of nucleases to slx4 in icl repair remains unclear . the slx4 complex is capable of processing holliday junctions ( hjs ) in  vitro ( fekairi et  al . , 2009 ; muoz et  al . , 2009 ; svendsen et  al . , hjs are four - way dna junctions at which two  chromatids are topologically intertwined . these structures arise during homologous recombination ( hr ) , a process required for repairing unscheduled double - strand breaks ( dsbs ) or damaged replication forks in mitotic cells . hjs are also key intermediates during meiotic recombination ( schwartz and heyer , 2011 ; west , 2009 ) . ultimately hjs must be removed to enable chromosome segregation , and two distinct modes of hj removal have been identified in mammalian cells ( schwartz and heyer , 2011 ) . the first pathway involves the dissolution of double hjs ( dhjs ) by the btr complex ( blm - topiii - rmi1-rmi2 ) . the coupled helicase and topoisomerase activities of blm and topiii , respectively , disassemble hjs , resulting exclusively in noncrossover products ( chaganti et  al . this pathway dominates in mitotic cells , possibly because minimizing crossovers lowers the incidence of loss of heterozygosity ( loh ) that would increase disease risk and impair organism fitness ( larocque et  al . , 2011 ) . alternatively , hjs can be resolved by nucleases ( schwartz and heyer , 2011 ; west , 2009 ) . depending on the symmetry of the cleavage , crossover or noncrossover products may occur . cells from bloom syndrome ( bs ) patients lacking blm show a large increase in the frequency of sister chromatid exchanges ( sces ) , which are thought to result from the crossovers generated by nucleolytic resolution of dhjs that escape dissolution ( chaganti et  al . , 1974 ; wechsler et  al . , , three nuclear hj resolving activities have been identified in mammalian cells : mus81-eme1 , slx1 , and gen1 ( bailly et  al . , 2010 ; fekairi et  al . , 2009 ; ip et  al . , 2008 ; muoz et  al . , 2009 ; svendsen et  al . , 2009 ; taylor and mcgowan , 2008 ; wechsler et  al . , 2011 ; west , 2009 ) . gen1 cleaves hjs symmetrically to produce nicked linear duplex products ( ip et  al . , 2008 ; rass et  al . , 2010 ) , whereas slx1 introduces a mixture of symmetric and asymmetric cuts across the junction ( fekairi et  al . , 2009 ; in contrast to gen1 and slx1 , mus81-eme1 does not cleave intact hjs efficiently , but prefers nicked junctions and recombination intermediates such as extended d - loop structures ( boddy et  al . , 2003 ; doe et  al . , 2002 ; gaillard et  al . , 2003 ; the elevated sce frequency in bs cells requires gen1 and mus81-eme1 ( wechsler et  al . , 2011 ) . however , although gen1 cleaves intact hjs efficiently in  vitro , mus81-eme1 does not . instead mus81-eme1 shows a strong preference for nicked hjs , suggesting that it might act on junctions that are subjected to prior nicking by a different nuclease . one possible candidate is slx1 because it can process intact hjs efficiently in  vitro . furthermore , slx1 and mus81-eme1 bind to the hth motif and the sap domain of slx4 , respectively ( fekairi et  al . , 2009 ; kim et  al . , 2013 ; svendsen et  al . , the close proximity of the slx4 sap and hth domains places slx1 close enough to mus81-eme1 on slx4 to suggest the possibility of cooperation between the two nucleases . in this sense , one function of slx4 in its capacity as a scaffold would be to facilitate serial processing of hjs , a possibility raised previously ( svendsen et  al . , 2009 ) . in this study , we investigated whether slx1 is involved in hj resolution and icl repair , and we tested potential cooperation with mus81-eme1 and the functional significance of binding of these nucleases to slx4 . there is currently no information on slx1 function in mammals . to study the in  vivo roles of slx1 , we disrupted the murine giyd2 ( slx1 ) gene by eliminating the transcription start site and the remainder of the first exon ( figure  s1a ) . gene disruption was confirmed by southern blotting and pcr ( figures s1b and s1c ) . both slx1 and slx1 mice were born at mendelian frequencies ( table s1 ) without overt morphological , developmental , or hematological defects . slx1 protein was undetectable in extracts of slx1 mouse embryonic fibroblast extracts ( mefs ) ( figure  1a ) or testis extracts ( figure  s1d ) by western blotting . the expression levels of slx4 , ercc1 , and mus81 proteins , however , were normal in slx1 mefs ( figure  1a ) . we also disrupted the murine btbd12 ( slx4 ) gene ( figure  s2a ) ; gene disruption was confirmed by southern blotting , pcr ( figures  s2b and s2c ) , and western blotting ( figure  s2d ) . the expression levels of slx4-associated proteins ercc1 and mus81 were normal in mefs from the slx4 mice but slx1 protein was undetectable by western blotting ( figure  1a ) . this suggests that slx4 regulates slx1 protein stability , and therefore slx4 null mice lack both slx1 and slx4 proteins . initially , no viable slx4 offspring were obtained from crossing slx4 heterozygotes . however , after backcrossing the heterozygotes five times , we obtained viable slx4 mice , albeit at sub - mendelian frequencies ( table s2 ) . slx4 mice were on average around 10%15% smaller than heterozygotes or wild - type mice at the age of 6  weeks ( data not shown ) . although mating slx4 males with slx4 females resulted in viable progeny , testes in males were on average 47% smaller than in wild - type mice at 10  weeks of age ( data not shown ) . smaller testis size is in line with two previous reports describing a hypomorphic slx4 mouse strain made by the european conditional mouse mutagenesis program ( eucomm ) ( crossan et  al . , 2011 ; it is worth noting that the eucomm mice were reported to exhibit phenotypes reminiscent of those seen in fa patients such as aplastic anemia ( crossan et  al . , 2011 ) , even though mouse knockouts of other fa genes did not recapitulate most of these phenotypes ( bakker et  al . , 2013 ) . however , we saw no evidence of an fa - like syndrome in the slx4 or slx1 mice we generated in this study ( data not shown ) . we next investigated if slx1 is involved in icl repair assessed by hypersensitivity to genotoxins that induce icls . as shown in  figure  1b , slx1 mefs are hypersensitive to icl - inducing agents such as nitrogen mustard ( hn-2 ) and mmc , and embryonic stem cells ( escs ) from slx1 mice were also hypersensitive to  mmc ( figure  s3a ) . the sensitivity of slx1 mefs to agents that induce icls was much less pronounced than slx4 mefs , probably because slx4 binds to several nucleases involved in icl repair in addition to slx1 . slx1 mefs and escs were not more sensitive to camptothecin ( cpt ) , ionizing radiation ( ir ) , hydroxyurea ( hu ) , or uv light than wild - type cells ( figures s3a and s3b ) . defects in the repair of icls often result in chromosome  abnormalities ( auerbach , 2009 ) . in this light , we observed a slight increase in the number of chromosome abnormalities such as chromatid breaks and radial structures in  slx1 mefs exposed to mmc compared with wild - type cells , and a much larger increase in slx4 mefs ( figure  s3c ) . we next tested if the nuclease activity of slx1 is required for cellular resistance to icl - inducing agents . to this end we mutated a highly conserved residue , glu79 , in the slx1  uri - type nuclease domain to alanine . this mutation abolished the activity of flag - tagged mouse slx1 immunoprecipitated from hek293 cells in the cleavage of a radioactively [ 5-p]-labeled hj with a core that could undergo a number of steps of branch migration , thereby presenting all possible dinucleotides at the point of exchange ( figure  1c ) ( muoz et  al . , 2009 ) . next , slx1 mefs were infected with viruses expressing untagged slx1 wild - type or e79a , or with empty virus ; mutation of e79 in slx1 did not affect interaction with slx4 ( figure  1d ) . as shown in figure  1e , wild - type slx1 , but not the e79a mutant , rescued the mmc hypersensitivity of slx1 mefs . together , these data provide evidence that slx1 is involved in repair of dna icls in mammals . , 2009 ; muoz et  al . , 2009 ; svendsen et  al . , we next investigated whether slx1 is required for hj resolution in  vivo using the elevated sce frequency observed in blm - depleted cells as readout ( figure  2a ) . to this end , we used shrna - expressing retroviruses to deplete blm from slx1 mefs ( figure  s4a ) ( sfeir et  al . , 2009 ) . as shown in figure  2b , blm protein was undetectable in mef extracts when cells were infected with retrovirus expressing the blm shrna , but not when empty virus was used . depletion of blm from wild - type mefs caused an increase in sce frequency from 8.3 sce per metaphase to 18 sce per metaphase ( figure  2c ) , but in two separate clones of slx1 mefs , the sce frequency after blm depletion was diminished to around 12 sce per metaphase . a similar defect in sce was observed in slx4 mefs ( figure  2c ) , in agreement with a previous analysis of bs cells depleted of slx4 ( wechsler et  al . the sce defect in slx1 mefs depleted of blm was rescued by expression of wild - type slx1 but not by the nuclease - inactive slx1 e79a mutant ( figure  2d ) . exposure of wild - type mefs to mmc caused a dose - dependent increase in sce frequency , but slx1 mefs were indistinguishable from wild - type cells in this regard ( figures 2e and 2f ) . in contrast , the mmc - induced increase in sce frequency was reduced by more than 30% in slx4 mefs ( figures 2e and 2f ) . thus , although slx4 contributes to the generation of sce during icl repair , slx1 does not appear to be involved . this could be due , in principle , to redundancy between slx1 and nucleases such as mus81-eme1 , as discussed below . the phenotypes associated with disruption of slx1 in mice described above are somewhat reminiscent of those reported for mus81 mice ( dendouga et  al . , 2005 ) . in both cases , the mice are viable and fertile with no gross abnormalities , but mefs are hypersensitive to icl - inducing agents . cells from both mice also show a higher than normal level of chromosome abnormalities after exposure to mmc . these observations , together with the physical proximity of slx1 and mus81-eme1 bound to slx4 , suggest that these two nucleases might act in the same pathway , perhaps cooperatively . in order to  test this possibility , we generated slx1 
 mus81 double knockout mice to enable epistasis analysis . these mice were born at mendelian frequencies ( table s3 ) , showing no developmental , morphological , or hematological abnormalities . moreover , the fertility of slx1 , mus81 , or slx1 mus81 littermates , or testis size in males , is not significantly different from wild - type mice ( data not shown ) . to study hj resolution , we isolated mefs from slx1 
 mus81 embryos , and as control we used mefs from single knockout embryos ( slx1 
 mus81 and slx1 
 mus81 ) . double heterozygous ( slx1 
 mus81 ) embryos were used as negative control ( figure  s4b ) . as mentioned earlier , slx1 had no effect on sces generated in mefs exposed to mmc ( figures 2e and 2f ) , and similar data were reported for mus81 cells ( mcpherson et  al . , 2004 ) . however , it was possible this is because slx1 acts redundantly with mus81-eme1 . however , as shown in figure  3a , the increase in sce frequency induced by mmc in cells lacking both mus81 and slx1 was indistinguishable from control cells ( slx1 
 mus81 ) and the slx1 or mus81 single knockouts . we also compared the mmc sensitivity of slx1 
 mus81 mefs with the respective single knockouts in clonogenic survival assays . as shown in figures 3b , the mmc hypersensitivity of mus81 cells was slightly more pronounced than slx1 cells , and slx1 
 mus81 cells were not more sensitive than the most sensitive of the single knockouts ( mus81 ) . similar results were obtained using alternative clones of mefs ( figure  s4c ) . taken together , these observations suggest that slx1 and mus81-eme1 are epistatic in terms of icl repair , at least judged by hypersensitivity to mmc , but they are not required for sce induced by icls in mefs . we went on to explore the possibility that slx1 and mus81-eme1 are also epistatic in the resolution of hjs . to this end , we depleted blm from mefs of various genotypes using shrna - expressing retroviruses ( figure  3c ) . depletion of blm from control mefs caused an increase in sce frequency from around 7 sce per metaphase to around 20 sce per metaphase . in slx1 or mus81 single knockout mefs , the sce frequency after blm depletion was diminished to around 10 sce per metaphase , and no further decrease was observed in slx1 
 mus81 double knockout cells leaving the residual levels of sce unaffected ( figure  3d ) . therefore , slx1 and mus81-eme1 are epistatic with regard to the resolution of hjs that escape dissolution , suggesting that they function in the same pathway . the data above suggest that slx1 and mus81-eme1 act together in hj resolution , and we next set out to test if this requires the tethering of the two nucleases to slx4 where they bind close together . the slx1-interacting region of slx4 has been localized to a small c - terminal fragment containing the hth motif ( figure  4a ) ( fekairi et  al . , 2009 ; svendsen et  al . , a 200 amino acid fragment containing the hth domain was subjected to saturated mutagenesis , followed by reverse yeast two - hybrid screening in order to find slx4 mutations that abolish interaction with slx1 ( data not shown ) . two such mutations were identified : a single point mutation in a highly conserved cys residue in the slx4 hth domain ( c1536r ) , and a small deletion mutant lacking six amino acids ( cys1536-thr1541 ; figure  4a ) . full - length slx4 bearing these mutations , or wild - type slx4 , was stably expressed in slx4 mefs . as shown in figure  4c , slx1 is undetectable in extracts of slx4 mefs , suggesting the stability of slx1 requires that it binds to slx4 . stable expression of wild - type slx4 restored normal levels of slx1 in slx4 mefs . however , the slx4 mutants c1536r or 1536 - 1541 , expressed at levels similar to wild - type slx4 , did not restore slx1 expression ( figure  4c ) . furthermore , no slx1 could be detected in slx4 immunoprecipitates from cells expressing these mutant forms of slx4 , but mus81 and ercc1 were present at normal levels ( figure  4c ) . these data indicate that the interaction of slx4 with slx1 is required for slx1 stability , and therefore cells expressing slx4 mutants that can not interact with slx1 are essentially null for slx1 expression . nonetheless , these mutants can be used to investigate if slx1 contributes to the roles of slx4 in dna repair . a previous study deleted the entire sap domain of slx4 to abolish interaction with mus81-eme1 , and the resulting slx4 deletion fragment could only partly rescue the mmc sensitivity of fancp cells ( kim et  al . , 2013 ) . however , even though in budding yeast slx4 does not interact with mus81-mms4 ( schwartz et  al . , 2012 ) , we found that deleting the entire sap domain of yeast slx4 perturbs its function ( data not shown ) . these data indicate that the sap domain of yeast slx4 is functionally important independent of mus81 interaction , and it is likely that the same is true of human slx4 . on this basis we sought to engineer more subtle changes in mouse slx4 that would abolish the interaction with mus81-eme1 . a fragment containing the sap domain of slx4 was subjected to saturated mutagenesis , reverse yeast two - hybrid and alanine scanning ( data not shown ) . these experiments revealed several mutations in slx4 that abolished interaction with mus81-eme1 : y1340a , l1348a , and a combination of e1351a and l1352a ( figure  4b ) . full - length slx4 bearing these mutations , or wild - type slx4 , were stably expressed in slx4 mefs . as shown in figure  4d , the slx4 y1340a , l1348a , and e1351a l1352a mutants were unable to interact with mus81-eme1 in coimmunoprecipitation experiments , but their ability to interact with slx1 and ercc1 was unaffected . we next examined whether the slx4 mutants that are unable to interact with slx1 or mus81-eme1 are able to rescue the defects observed in slx4 cells , starting with analysis of icl repair . as shown in figure  5a , the slx4 c1536r and 1536 - 1541 mutants , that are incapable of interacting with slx1 , only partly rescued the mmc hypersensitivity of slx4 mefs . the residual sensitivity of these cells was similar to that of slx1 cells ( figure  1b ) , consistent with a failure of c1536r and 1536 - 1541 slx4 mutants to restore slx1 expression in slx4 cells ( figure  4c ) . on the other hand , the slx4 l1348a and e1351a l1352a mutants , both of which fail to interact with mus81-eme1 , were able to fully rescue the mmc hypersensitivity of slx4 mefs ( figure  5b ) . referring to the slx4 c1536r mutant hereafter as slx4 and the slx4 e1351a l1352a as  slx4 , we combined these mutations to generate slx4 ( figure  s5 ) , and found that this compound mutant restored mmc hypersensitivity of slx4cells to the same degree as the slx4 mutant ( figure  5c ) . these data suggest that mmc hypersensitivity of cells lacking slx4 is at least partly due to lack of slx1 and that the interaction of slx4 with mus81-eme1 is not required for icl repair in mefs . we went on to test if the slx4 and slx4 mutants are defective in hj resolution . as shown in figure  5d , the elevation in sce frequency triggered by blm depletion is diminished in slx4 mefs compared with wild - type mefs . this defect is rescued by expression of wild - type slx4 , but not by the slx4 , slx4 mutants or the slx4 compound mutant . therefore , it is likely that the binding of both slx1 and mus81-eme1 to slx4 is essential for hj resolution . in contrast , the binding of slx1 , but not mus81-eme1 , is important for icl repair in mefs ( figure  5c ) . these data suggested strongly that the role of slx1 and mus81-eme1 in icl repair is independent of hj resolution at least in cells from mice , and we set out to investigate this possibility . the data above show that slx1 and mus81-eme1 are both involved in hj resolution , judged by measuring the increased sce frequency caused by blm depletion . however , slx1 and mus81-eme1 are both capable of cleaving a range of branched dna structures besides hjs in  vitro . this consideration , together with the observation that blm can act on a similar range of branched substrates ( mohaghegh et  al . , 2001 ; van brabant et  al . , 2000 ) , raised the possibility that the sces in blm - depleted cells arise from nucleolytic processing of structures other than four - way junctions . in an attempt to investigate this possibility , we set out to see if the cleavage of hjs by slx1 and mus81-eme1 is physiologically relevant . with this in mind , we mutated the slx1 nuclease domain ( r38a e79a ) to inactivate it and fused this slx1 derivative , which should maintain the ability to bind slx4 , to an archaeal resolving enzyme hje . this enzyme was reported to have a marked preference for hjs compared with other resolvases ( kvaratskhelia and white , 2000 ) . we next asked if the resulting fusion protein , referred to as hje - slx1 ( figure  6a ) , can rescue defects associated with loss of slx1 . we used an analogous fusion construct containing a catalytically inactive d39a hje mutant as a negative control . we confirmed that flag - tagged versions of the hje - slx1 fusion protein , and the catalytically inactive derivative , bind to slx4 in coimmunoprecipitation experiments in hek293 cells ( figure  s6b ) . we used a four - way junction with a 12  bp homologous core ( hj ) , a replication fork ( rf ) analog ( a three - way junction where one strand is discontinuous at the junction ) , and a 5 flap . reaction products were separated by gel electrophoresis under denaturing conditions . as shown in figure  6b , both slx1 and the hje - slx1 fusion exhibited strong cleavage activity on the four - way junction substrate . consistent with previous reports , slx1 cleaved the four - way junction at multiple positions ( fekairi et  al . , 2009 ; muoz et  al . , 2009 ; svendsen et  al . , 2009 ) ; the hje - slx1 fusion protein also cleaved at several points , but these were different from slx1 ( figure  6b ; figure  s6a ) . nonetheless , nondenaturing gel electrophoresis showed that hje - slx1 introduced symmetric cleavages within the homologous core of the hj , resulting in the production of linear duplex dna as required for the productive resolution of the junction ( figure  6c ) . the hje - slx1 derivative , which is inactivated by the d39a mutation , showed no detectable activity toward the hj substrate ( figure  6b ) . although all of the flag immunoprecipitates exhibited some nonspecific activity toward the rf substrate ( figure  6b , asterisk ) , a weak slx1-specific cleavage product was observed in  flag - slx1 immunoprecipitates that was not present in slx1 e79a precipitates ( figure  6b , arrowhead ) . this product was not observed in hje - slx1 precipitates , indicating that this fusion protein can not cleave replication forks . the 5 flap substrate was cleaved by flag - slx1 one nucleotide before the junction , and by contrast , the hje - slx1 fusion exhibited only very weak activity three nucleotides from junction ( figure  6b ; figure s6a ) . these experiments demonstrated that hje - slx1 binds to slx4 and acts as a potent hj resolving enzyme with restricted substrate specificity compared with slx1 . we next examined whether the hje - slx1 fusion could restore the cellular functions perturbed in cells lacking slx1 . untagged versions of hje - slx1 and the inactive d39a derivative were stably expressed in slx1 mefs ( figure  s6c , left panels ) . cells were then depleted of blm . strikingly , the hje - slx1 fusion protein , but not the d39a version , restored sce frequency in blm - depleted slx1 mefs similar to wild - type slx1 ( figure  6d ) . expression of hje - slx1 in cells that were not depleted of blm had no apparent effect on sce levels ( figure  6d ) . the hje - slx1 fusion and the inactive d39a derivative were also stably expressed in slx1 
 mus81 mefs ( figure  s6c , right panels ) . this experiment revealed that hje - slx1 but not the d39a version restored sce frequency after blm depletion back to wild - type levels ( figure  6e ) . taken together , these data strongly suggest that the formation of sce in blm - depleted cells , which was shown to require binding of slx1 and mus81-eme1 to slx4 , results from the resolution of hjs . finally , we tested the ability of the hje - slx1 fusion protein to reverse the icl repair defects in cells lacking slx1 . intriguingly , the hje - slx1 fusion protein was incapable of rescuing the mmc hypersensitivity of slx1 mefs in contrast to wild - type slx1 ( figure  s6d ) . these data strengthen the notion that the role of slx1 in icl repair in mefs involves cleavage of dna structures other than hjs . in this study , we demonstrate that slx1 is involved in cellular responses to dna damage in mammals . consistent with its reported ability to process hjs in  vitro ( fekairi et  al . , 2009 ; muoz et  al . , 2009 ; svendsen et  al . , 2009 ) , we found that slx1 , like mus81-eme1 , plays a major role in the nucleolytic resolution of hjs in  vivo . slx1 and mus81-eme1 are both required for resolving the majority of hjs that escape dissolution by blm , and they act epistatically in this regard . we also found that the tethering of slx1 and mus81-eme1 to slx4 , where they bind in close proximity , is essential for the ability of these nucleases to promote hj resolution . the simplest interpretation of these findings is that slx1 and mus81-eme1 act cooperatively in the processing of hjs , as previously postulated by others ( svendsen et  al . , 2009 ) . in this model slx1 acts on intact hjs to create nicked junctions , which are then cleaved by mus81-eme1 ( boddy et  al . , 2003 ; doe et  al . , 2002 ; svendsen et  al . , 2009 ; whitby et  al . , 2003 ) . close juxtaposition of slx1 and mus81-eme1 on slx4 might position the respective active sites to favor coordinated first - strand cleavage by slx1 and second - strand cleavage by mus81-eme1 . this idea might help to solve the longstanding dilemma of how mus81-eme1 participates in hj resolution in  vivo when the recombinant enzyme only weakly cleaves intact hjs in  vitro compared with nicked hjs ( boddy et  al . , 2003 ; doe et  al . , 2002 ; whitby et  al . , the model whereby slx1 and mus81-eme1 act together in hj resolution is based on measuring sce frequency upon blm depletion . but do the sces observed in blm - depleted cells result from the cleavage of hjs ? it is possible that the sces resulting from depletion of blm reflect instead the cleavage of branched dna structures other than hjs . this is a particular concern because blm , like mus81-eme1 and slx1 , is active toward a range of branched dna structures in  vitro ( mohaghegh et  al . , 2001 ; van brabant et  al . , 2000 ) . furthermore , the cleavage of the precursors to hjs such as extended d - loop structures can produce crossovers that would manifest as sces ( osman et  al . , thus , it is possible that the sces in blm - depleted cells occur as a result of the cleavage of  recombination structures other than hjs . however , the demonstration that the hje - slx1 fusion protein with increased specificity for hjs can reverse the sce defect in slx1 and slx1 
 mus81 cells strongly suggests that sce formation  in  blm - depleted cells reflects hj cleavage by slx1 and mus81-eme1 . in this study we demonstrate that slx1 is involved in icl repair in mammalian cells , and similarly to hj resolution , slx1 appears to act epistatically with mus81-eme1 in icl repair . however , several observations suggest that the epistatic interaction between slx1 and mus81-eme1 in icl repair does not involve the cleavage of hjs , at least in mice . first , the sces formed in mmc - treated mefs do not require slx1 or mus81-eme1 , unlike the sces observed in blm - depleted cells . second , whereas hj resolution in blm - depleted mefs requires binding of both slx1 and mus81-eme1 to slx4 , cellular resistance to mmc requires binding of slx1 but not mus81-eme1 . third , the hje - slx1 fusion protein , which is much more specific for hjs than  slx1 , can rescue the sce defect in slx1 and slx1 
 mus81 mefs depleted of blm , but it can not rescue the mmc hypersensitivity of these cells . these observations suggest that the role of slx1 and mus81-eme1 in the repair of icls involves the processing of structures other than hjs . mus81-eme1 is known to act at the  unhooking  step of icl repair , a process that involves incisions on either side of the icl that produce dsbs ( hanada et  al . however , dsb induction in cells exposed to icl - inducing agents does not require slx1 ( data not shown ) . therefore , the epistatic interaction of slx1 and mus81-eme1 in icl repair is independent of dsb induction . one possible explanation is that slx1-slx4 processes recombination intermediates during the repair of the dsbs generated by the pool of mus81-eme1 that is not bound to slx4 . it is clear that the dominant mode of hj removal in mitotic cells  at least those arising spontaneously  involves blm , and hj resolution by nucleases acts to cleave junctions that escape dissolution . the blm pathway has been supposed to minimize the risk of loss of heterozygosity , since this pathway generates noncrossover outcomes exclusively ( larocque et  al . , 2011 ) . however , in meiotic cells crossover formation is necessary and should therefore involve nucleolytic hj resolution . since slx1 and mus81-eme1 resolve the majority of hjs that escape dissolution in mitotic mammalian cells , one might predict that they might dominate in meiosis . however , although mus81 mice show meiotic defects , they are fertile , suggesting that other nucleases can mediate hj processing in the absence of mus81-eme1 ( dendouga et  al . , 2005 ; this is unlikely to be slx1 , since the fertility and gonad size of slx1 , mus81 , or slx1 mus81 mice generated in this study were not significantly different from wild - type mice ( data not shown ) . since slx4 mice exhibit subfertility and hypogonadism , but slx1 mus81 do not , the third slx4-associated nuclease , xpf - ercc1 , might be involved in meiotic hj resolution . in this light , recent studies in nematodes found that xpf / blm and slx1/mus81-eme1 act on parallel pathways in worm meiosis ( agostinho et  al . , 2013 ; oneil et  al . , 2013 ; saito et  al . , moreover , slx4 and xpf - ercc1 are important for crossover generation in drosophila ( yildiz et  al . , 2002 ) . it will  be interesting to test if xpf - ercc1 is involved in hj resolution in mammals . however , since slx4 mice can generate offspring , it must be that nucleases that do not associate with slx4 can resolve hjs in the absence of slx4 complex components . . it will be interesting to make the relevant combinations of gene knockouts in mice to investigate this . given the importance of meiotic hj resolution in heredity , and in preventing defects in chromosome segregation , it will be vitally important to decipher the precise mechanisms underlying the resolution of meiotic hjs . the protocol was adapted from a previous report ( sfeir et  al . , 2009 ) . briefly , viruses expressing a blm - specific shrna were produced by cotransfecting 293 t cells with psuper.retro.puro ( open biosystems ) expressing the blm target sequence 5-gga cct gct gga aga ttt a-3 and with pcmv - vsv - g and pcmv - gag - pol . mefs were infected four times in 6  hr intervals with virus - containing supernatant supplemented with 8  g / ml polybrene . twenty - four hours after the last infection , mefs were selected with 3  g / ml puromycin . cells were treated for 24  hr with 10  m brdu before replacing the medium with fresh brdu and the indicated dose of mmc for an additional 24  hr . when sce analysis was performed after blm knockdown , cells were selected for the uptake of virus in the presence of 3  g / ml puromycin 24  hr after the last infection . mefs were treated with two cycles of brdu as described above ( figure  s4a ) . at 6 hr before the end of the second brdu cycle , nocodazole was added at a final concentration of 0.15  g / ml . cells were harvested and prepared for metaphase spreads . spreads were stained with hoechst 33258 , followed by uv irradiation and staining with giemsa according to standard protocols . fifty spreads were analyzed for each condition on a deltavision wide - field deconvolution microscope ( olympus ) .
summaryholliday junctions ( hjs ) are x - shaped dna structures that arise during homologous recombination , which must be removed to enable chromosome segregation . 
 the slx1 and mus81-eme1 nucleases can both process hjs in  vitro , and they bind in close proximity on the slx4 scaffold , hinting at possible cooperation . however , the cellular roles of mammalian slx1 are not yet known . here 
 , we use mouse genetics and structure function analysis to investigate slx1 function . disrupting the murine slx1 and 
 slx4 genes revealed that they are essential for hj resolution in mitotic cells . 
 moreover , slx1 and mus81-eme1 act together to resolve hjs in a manner that requires tethering to slx4 . 
 we also show that slx1 , like mus81-eme1 , is required for repair of dna interstrand crosslinks , but this role appears to be independent of hj cleavage , at least in mouse cells . 
 these findings shed light on hj resolution in mammals and on maintenance of genome stability .