, it creates a transient physical linkage between homologous chromosomes to ensure accurate segregation and to facilitate genetic diversity in the process . in mitotic cells
, recombination provides a mechanism for the repair of dna double - strand breaks and facilitates the repair of interstrand crosslinks and dna lesions arising during replication .
the central intermediate species of recombination is the four - way ( holliday ) junction ( holliday , 1964 ) , in which four dna helices are temporarily connected by strand continuity .
processing of such junctions is a key event that can occur by dissolution or resolution .
dissolution involves translocation of two junctions toward each other by blm helicase followed by decatenation mediated by topoisomerase iii ( cejka et  al . , 2010 , ellis et  al . , 1995 , wu and hickson , 2003 ) .
by contrast , resolution involves the action of nucleases that are targeted to the structure of the four - way junction .
a number of junction - resolving enzymes from bacteria and their phages , archaea , and yeast mitochondria have been well characterized ( reviewed in dclais and lilley , 2008 ) .
these enzymes selectively bind four - way dna junctions in dimeric form with high affinity , recognizing and manipulating the junction structure , and introducing symmetrical , bilateral cleavages that result in productive resolution .
two main junction - resolution activities have been identified in eukaryotic cells , both unrelated to those from lower organisms .
the first was gen1 , identified by west and coworkers after a long search ( elborough and west , 1990 , constantinou et  al .
, 2001 , constantinou et  al . , 2002 ) and isolated through extensive biochemical fractionation of hela cells ( ip et  al . , 2008 ,
gen1 was also isolated from budding yeast ( ip et  al . , 2008 ) ( as yen1 ) and caenorhabditis elegans ( bailly et  al . , 2010 ) .
the other main activity arises from the combination of slx1-mus81-eme1-slx4 proteins ( agostinho et  al . , 2013 , andersen et  al . , 2009 , fekairi et  al . , 2009 , muoz et  al . , 2009 ,
at least one of these activities must be functional to maintain cell viability , as gen1 and slx4 are synthetically lethal in human cells due to dysfunctional mitosis resulting from unprocessed junctions ( garner et  al .
the meiotic phenotype of mus81 fission yeast is restored by ectopic expression of human gen1 ( lorenz et  al . , 2010 ) , and
the budding yeast ortholog yen1 is required to resolve persistent dna junctions during meiosis when mus81 is deleted ( matos et  al . , 2011 ) .
gen1 is a member of a superfamily of structure - selective nucleases ( grasby et  al . , 2012 ) .
these include the enzymes fen1 that acts on various flap and double - flap structures ( ceska et  al . , 1996 ,
( orans et  al . , 2011 ) and xpg ( rad2 in yeast ) that acts in nucleotide excision repair ( mitus et  al . , 2014 ) .
this contains a relatively flat platform of dimension 70   30   , based upon a central seven - strand twisted  sheet flanked on both sides by a total of 15  helices , reminiscent of an elaborated rossmann fold .
all three proteins bind a dna duplex ( in fen1 this is the helix with the 5 flap strand ) , with a common element ( termed h2th ) that contacts the backbone of the uncleaved strand . in fen1 , a second dna duplex ( that with the 3 flap and connected to the duplex with the 5-flap by the continuity of the uncleaved strand ) is bound , such that the two axes are virtually perpendicular .
the active site is centrally located on the platform and comprises a number of conserved carboxylate residues around the - interface that coordinate one or two metal ions in the crystal structures .
mechanistic studies on phage t5 fen indicate that at least two metal ions participate in the hydrolysis reaction ( syson et  al . , 2008 )
. some functionally important helices project above the platform . in all cases , the  helix immediately c - terminal to the second section of  sheet ( counted from the n terminus ) projects above the  sheet region to abut the end of the dna duplex , splaying apart the strands
this helix directs a conserved tyrosine toward the dna in fen1 ( tsutakawa et  al . , 2011 ) .
a second section of polypeptide observed in fen1 and exo1 is directed above the platform , comprising 40 amino acids in two  helices .
this is termed the helical arch , through which passes the single strand of the substrate thereby selecting substrates with such features .
although sequence homology indicates that gen1 will contain a number of features in common with fen1 , exo1 , and xpg , we would anticipate that it would have to differ from the other family members in a number of key respects .
given that gen1 resolves a four - way dna junction , it must act in dimeric form , in common with all known junction - resolving enzymes ( dclais and lilley , 2008 ) , in agreement with our recent analysis ( freeman et  al . ,
since the strands of a four - way junction are base - paired and covalently continuous , it would not require the helical arch ,  so this feature would probably be extensively modified in  gen1 .
although the active site would be expected to be conserved in gen1 , the key question of interest is how the enzyme is selective for dna junctions . to answer this
studies of gen1 from human cells have shown that the n - terminal section acts in dimeric form to resolve four - way dna junctions ( rass et  al . , 2010 ) .
however , all fragments of the enzyme have been found to be poly - disperse and fail to form discrete complexes with junctions .
in contrast , we found that the orthologous enzyme from the thermophilic fungus chaetomium thermophilum was very well behaved ( freeman et  al . , 2014 ) .
in free solution the protein exists primarily in monomeric form , but binds to dna junctions as a discrete dimer to generate bilateral cleavage by accelerating second strand cleavage .
the biochemical properties of this enzyme conform closely to those established for the junction - resolving enzymes as a class ( dclais and lilley , 2008 ) .
we have now solved a crystal structure of active gen1 from c.  thermophilum bound to the dna resulting as the product of cleavage .
the n - terminal 1487 wild - type amino acid sequence of c.  thermophilum gen1 protein ( hereafter referred to as ctgen1 ) with a c - terminal six - histidine tag was expressed in escherichia coli , using normal and selenomethionine - containing medium .
purified ctgen1 was mixed in equimolar quantities with a four - way dna junction based on the well - characterized junction 3 ( duckett et  al . , 1988 ) and comprising 15  bp in each helical arm .
equal volumes of dna and protein were mixed in a final concentration of 100  mm hepes ( ph 7.5 ) , 2  mm mgcl2 , 20% peg10000 , and incubated with the same buffer using hanging drop vapor diffusion at 7c .
experimental phasing was achieved by single - wavelength anomalous dispersion using the selenomethionine - substituted ctgen1 ( pdb : 5co8 ) .
some crystals were soaked in 1  mm mncl2 solution to exchange mg with mn ions ( pdb : 5cnq ) .
the two crystal forms were solved at a resolution of 2.5 and 2.6   , respectively .
the presence of mgcl2 in the crystallization solution induced cleavage of the four - way dna junction by ctgen1 . in principle ,
resolution cleavage could lead to four possible products , resulting from cleavage of either pair of two opposing strands ( i.e. , either strands b and r or strands h and x , figure  1a ) .
however , our previous biochemical experiments show there is a very strong bias toward cleavage on the h and x strands of junction 3 ( freeman et  al . , 2014 ) , giving just two products ( in  which the b and r strands remain intact )
. electrophoretic analysis of the dna contents of our crystals ( figure  s1a ) shows that the crystallization has selected one of the two products , i.e. , that with the intact r strand .
the junction used in crystallization trials was assembled from four 30 nt strands that formed four 15  bp helices and had an asymmetric core sequence that precluded branch migration .
given that ctgen1 cleaves strands 1 nt 3 of the junction , the crystallized product should comprise a 14  bp helix ( the 3 14 nt of the h strand and the 5 14 nt of the r strand ) and a 15  bp helix ( the 3 15 nt of the r strand and the 5 15 nt of the x strand ) , with a mismatch between the two helices comprising nt 15 of the r strand and nt 16 of strand x ( figure  1a ) .
the crystals belong to the p3121 space group , in which the asymmetric unit contains one ctgen1 monomer and a duplex of dna with 14 and 15 nt strands ( 14  bp with a 3 overhang ) that adopts a standard b - form helix with no distortion .
the 5 end of the 14 nt strand is located in the active site of the enzyme , indicated by the presence of a bound mg ion ( figure  1b ) .
how does this asymmetric unit arise from the crystallized product dna that includes a 30 nt strand ?
the explanation must be that the two duplexes of the product occupy crystallographically equivalent positions within the lattice .
the structure of the asymmetric unit arises naturally from resolution cleavage of the h  strand , which produces a 14  bp helix with the 5 end of the 14 nt h strand in the ctgen1 active site and a mismatched 3 nucleotide on the r strand . within the crystal lattice
, the other half of the product molecule is in a crystallographically equivalent position with respect to another ctgen1 monomer , with the 5 end of its x strand located in the active site of the second monomer ( figure  1c ; movie s1 ) . as a result ,
contributions from both halves of the product dna become averaged in the electron density map and have been modeled with 50% occupancy ( figure  s1b ) .
the second dna helix should comprise a 15  bp duplex with a 1 nt 3 overhang .
however , its end is frayed by insertion into the active site of the second ctgen1 monomer and the 5  x nucleotide is not seen . neither is the overhanging 3 nt of the x strand , thus only 14 nt of the x strand are observed , making it equivalent to the h strand in the structure .
the axes of the two duplexes forming a product molecule bound to one ctgen1 subunit are virtually perpendicular . in the crystal lattice
, a second monomer brings together the distal ends of duplexes from two adjacent products at a 90 angle ( yellow monomer in figure  s1b and movie s1 ) .
the 5 end of the r strand of one product is juxtaposed with the 3 end of the r strand of an adjacent product such that the r strand appears quasi - continuous between the two product molecules .
inspection of an fo - fc electron density map calculated from a model lacking the central phosphate reveals clear density corresponding to a phosphate connecting the two halves of the 30 nt long r - strand with partial occupancy ( figure  1d ) , in agreement with the expected averaging between central and distal ends of the duplexes .
the head - to - tail gen1-bound products form parallel chains running through the crystal lattice in three orientations related by the trigonal symmetry ( movie s2 ) .
however , these parallel chains are not all in register ; the products in one strand could align with those of an adjacent strand , or they could be offset such that the h strands in one chain align with the x strands of an adjacent strand .
this is because all of the contacts within the lattice are protein - mediated , and gen1 binds each half of the product in an equivalent manner .
this lack of alignment results in the averaging of the dna sequences in the electron density map .
we have , therefore , associated the 30 nt strand as one covalently continuous strand within the complex and will represent it in that manner hereafter . thus the functional unit contains a product of resolution ( figure  1e ) , comprising a 30  nt r strand , the 14 nt 3 section of the h strand ( referred to subsequently as 3h ) and the 16 nt 5 section of the x strand ( 5x ) of which the first and last nucleotides are not visible in the electron density .
the trajectory of the dna is strikingly similar to that bound to human fen1 ( hfen1 ) ( tsutakawa et  al . , 2011 ) .
the dna from the two complexes can be superimposed with a root - mean - square deviation ( rmsd )  = 2.88   ( figure  2d ) .
we have fitted 84% of the amino acids in the calculated electron density maps , modeling all the secondary structure except for a  number of disordered loops and the c - terminal 22 amino acids . 
ctgen1 is an approximate hemi - ellipsoid of dimensions 80   30   30   , broadening at one end to 40   ( figure  2a ) .
the shape can be likened to a rowing boat , with the dna bound on one edge of the relatively flat surface .
it is constructed around a central seven - strand  sheet that is parallel except for 7 .
it is flanked on both sides by  helices , including the four - helix bundle comprising 4 and 5 together with 14 and 15 that buries an area of 1,256   .
the connectivity of helices and sheet is in most respects identical to that of the fen1 family members .
the most prominent difference between ctgen1 and hfen1 is that the two helices forming the helical arch in the latter are not observed in ctgen1 .
these helices would lie between 3 and 5 in ctgen1 ; if they adopted a regular structure they would be visible , yet that section can not be observed in the electron density map consistent with it being unstructured and flexible .
moreover , there are only 12 amino acids located between 3 and 5 in ctgen1 whereas the helical arch of hfen1 comprises 39 amino acids .
if this section is excluded from hfen1 , then it and the observed sections of ctgen1 from the n terminus to 15 superimpose with an rmsd  = 2.07   ( figure  2c ) , showing that the two proteins are closely related in structure .
the c - terminal section of ctgen1 contains a three - strand antiparallel  sheet ( 810 ) and four  helices .
this section fills the wider end of the structure ( the stern in the rowing boat analogy ) and has no counterpart in the other fen1-xpg family members . submission to the dali server (
holm and rosenstrm , 2010 ) indicates that the structure is similar to a series of chromobox homology proteins .
the two sections of ctgen1 are connected by a disordered 15 amino acid peptide not observed in the electron density .
the c terminus of ctgen1 lies at the end of 19 , directed away from the protein . in the full - length gen1
we find that we can fuse additional sections of proteins c - terminal to residue 530 without loss of enzyme activity .
by contrast , the n terminus of ctgen1 is located at a dna - protein interface and only 4   from the active site .
any modification of the n terminus results in complete loss of activity ( data not shown ) . within the functional unit , each ctgen1 monomer
is bound to one product dna molecule , derived from two arms of the four - way junction as the product of resolution cleavage ( figure  1a ) .
the dna - binding face of ctgen1 contains a number of lysine and arginine residues , forming a predominantly electropositive ( i.e. , basic ) track especially at the points of backbone contact ( figure  3a ) , burying a surface area of 1,394   .
altogether , both dna helices are held by multiple points of attachment to both strands ( figure  s3 ) , constraining their relative perpendicular orientation in the complex . the cleaved dna helix ( the r arm of the product , i.e. , the helix containing the 5 end of the 30 nt r strand ) is bound at the  bow  end of the ctgen1 molecule . a complete turn of dna is bound along one edge of the flat platform generated by helices 1 , 9 , 10 , and 12 and their connecting loop regions .
helices 9 plus 10 correspond to the h2th motif observed in hfen1 ( tsutakawa et  al . , 2011 ) .
in addition , helix 10 of the h2th motif is oriented directly at the backbone , with its n - terminal end 4   from a phosphate group of the non - cleaved strand such that its charge is partially neutralized by the positive pole of the helix dipole ( figure  3b ) .
helix 3 is positioned at the end of the cleaved helix , incompatible with the continuation of double - stranded helical geometry , and helix 4 is similarly positioned at the end of the uncleaved helix ( figure  3c ) . the unit comprising 3 , 4 , and the connecting coil region is functionally equivalent to the helical wedge of hfen1 .
the phenyl side chain of f44 on 3 is stacked with the unpaired nucleobase at the hinge of the unbroken r strand ; this position aligns with y40 in hfen1 , where it has a similar function .
the strand connecting the two duplexes passes between the helical wedge 3 and 4 and the loop between 6 and 7 .
the uncleaved duplex ( the x arm of the product , containing the  3 end of the unbroken r strand ) is located in a strongly electropositive cleft , with backbone contacts between the r strand and 4 and 15 and the 5x strand with the three - strand  sheet ( figure  3d ) .
these contact 8  bp in total , on one face of the dna helix .
the c - terminal section of ctgen1 not found in hfen1 contacts an additional half - turn of dna , thus adding substantially to the contacts on that arm .
the 5 end of the 3 h strand ( i.e. , the site of nucleolytic cleavage by the resolving enzyme ) is directed down into a strongly electronegative cavity near the center of the enzyme that we assign to be the active site of the enzyme ( figure  4 ) .
the cavity contains six conserved acidic amino acids , contributed by the n - terminal ends of 6 , 7 , and the c - terminal ends of 2 and 3 .
metal ions are bound in the middle of this cluster of acidic side chains . for the original mg crystals , a single bound ion ( m2 )
was observed ( figure  4a ) , but after soaking the crystals with mncl2 , two bound metal ions were observed ( figures 4b and s4 ) .
metal ion m2 is 2.1   from the carboxylate groups of d141 and d143 , while m1 is 2.2   from the carboxylate group of e122 , 4.1   from that of d38 and d79 , and 4.7   from e120 .
substitution of each acidic residue individually to alanine ( table s1 ) shows that while dna binding is almost unaffected by removal of the carboxylate groups , cleavage activity is impaired for each mutant , in most cases by orders of magnitude .
it is very likely that the metal ions adjust their position within the pocket during the course of binding of the four - way junction , as the phosphodiester group becomes directly coordinated and the reaction progresses through the cleavage of the two strands . in the standard two - metal ion model of phosphoryl transfer reactions ( steitz and steitz , 1993 ) ,
the metal ions serve to activate the water nucleophile , stabilize the anionic transition state and position the reactants .
nucleases operating such a mechanism frequently use a positively charged side chain to stabilize the transition state , exemplified by the active site lysine of the junction - resolving enzyme t7 endonuclease i ( dclais et  al . , 2001 ) .
there are a number of candidates for this role within the unobserved region between 3 and 5 .
examination of the crystal lattice reveals that protein - protein interaction between ctgen1 monomers brings two bound dna products into close proximity as though forming a four - way junction ( figures 5a , 5b , s5a , and s5b ; movie s2 ) .
the two ctgen1 molecules interact primarily by the ends of alpha helices 4 , 5 , and 14 ( figures 5c , 5d , and s5c ) and their associated loops , burying a surface area of 530   .
the interaction generates an almost coaxial alignment of the uncleaved dna helices , while the cleaved helical arms rotate toward each other on the major groove side such that they include an angle of close to 90. an axis of 2-fold symmetry bisects the plane defined by the axes of these two helices and passes through the center of , and is normal to , the coaxially aligned helices . within this complex ,
it is possible to reconnect the 5 ends of the h strands with the 3 ends of the x strands to generate a covalently intact four - way junction ( figure  5e ; movie s3 ) .
this requires the base pair located at the junction - proximal end of the uncleaved helix to be broken and unstacked , the nucleotide at the 3 end of the x strand to be rotated around toward the h strand and the ( unobserved ) 16th nucleotide of the x strand to be modeled to make the phosphodiester linkage ( figure  s6 ) .
these two nucleotides lie close to and may stabilize the disordered amino acids between 3 and 5 .
we have investigated the overall shape of  the complex of ctgen1 bound to an intact four - way dna junction in solution using comparative gel electrophoresis ( lilley , 2008 ) .
this method was originally used to determine the structure of the four - way dna junction in free solution ( duckett et  al . , 1988 ) and has been extended to study the shape of junctions bound to junction - resolving enzymes ( duckett et  al . , 1995 , giraud - panis and lilley , 1998 , phler et  al . , 1996 , white and lilley , 1996 , white and lilley , 1997 ) . in this method
, we compare the electrophoretic mobility of the six possible forms of a junction with two long ( here 40  bp ) and two short ( 14  bp ) arms .
the global shape of the junction can be deduced from the symmetry and pattern of the relative mobilities of the different species , since the relative mobility of species increases with the angle included between the long arms .
six long - short arm species of junction 3 comprising all combinations of two long and two short arms were constructed , each from four synthetic radioactively [ 5-p]-labeled dna strands .
a fraction of each junction was incubated with a molar excess of ctgen1 to form an enzyme - junction complex .
these were then loaded on to a 5% polyacrylamide gel and electrophoresis was performed under non - denaturing conditions in the presence of 50  mm nacl and 2  mm cacl2 ; these conditions induce folding of the junction but inactivate the nuclease activity of the enzyme .
the junctions in complex with ctgen1 migrate significantly more slowly than free junctions and exhibit a completely different pattern of relative mobility compared to a free junction ( duckett et  al . , 1988 ) .
the pattern of the complexes comprises five species migrating at an equal , slower rate , with just one species ( hx ) of significantly faster mobility ( figure  6 ) .
species rx corresponds to that seen in the crystal , and thus the long r and x  helices should include 90. species hr has virtually identical mobility so should also include 90. in euclidian geometry , if rx  = hr  = 90 this requires species hx ( i.e. , the fast - migrating species ) to include 180. species bh and bx also have closely similar mobility to rx , and species bh is expected to be equivalent to rx as the other product species , not observed in the crystal .
so once again this requires hx to include 180. this only leaves the angle br ( i.e. , that between the cleaved helices ) undetermined . while that species migrates as a slightly less well - defined band , it is evidently similar in mobility to the other 90 species , so these helices should also be close to mutually perpendicular . thus , the only model that is compatible with all the angular constraints is that shown in figure  6 .
this can be visualized by laying the junction in a plane with the arms pointing to the four corners of a square and then lifting the b and r arms ( i.e. , the helices that are cleaved by ctgen1 ) up so that they too  become mutually perpendicular .
in fact , all the helical arms are mutually perpendicular except for the diagonally opposed arms h and x that must be approximately  coaxial .
the model emerging from the solution study is in complete agreement  with the structure observed for the dimeric complex with two product molecules in the crystal ( figure  5e ) . in altering the conformation of the junction from the stacked x - structure in the absence of protein
, ctgen1 must disrupt the coaxial stacking of the arms , and more extensive disruption of base - pairing is expected if the protein bound junction has the structure presented in figure  5 .
central distortion of junction structure has been observed in a number of complexes with other junction - resolving enzymes ( dclais et  al . , 2003 ,
we have previously used two methods to detect base unstacking and disrupted base - pairing ; thymine bases become susceptible to electrophilic attack at the 5,6 double bond by permanganate ion , and 2-aminopurine bases exhibit enhancement of fluorescence .
we studied the reactivity of the thymine bases in the four strands of junction 3 .
two versions of the junction were individually radioactively [ 5-p]-labeled on either the h or x strand ( that have thymine nucleotides at the point of strand exchange ) and reacted with 1  mm kmno4 for 2  min at 25c in the presence or absence of an excess of ctgen1
. reacted thymine nucleotides were detected by cleavage with piperidine and separation of products by gel electrophoresis and phosphorimaging ( figure  7a ) .
the results show that just a single thymine base of the junction is reactive in the ctgen1 complex , but not the free dna , on each of the h and x strands .
these two thymine bases are present immediately at the point of strand exchange of the junction , and their reactivity is consistent with a disruption of the structure at the center of the junction on binding ctgen1 .
we analyzed the fluorescent intensity of versions of junction 3 in which a chosen adenine nucleotide was replaced by 2-aminopurine ( 2-ap ) .
the two adenines either side of the point of strand exchange on the r strand were individually substituted , as were those 3 nt 5 and 3 to the point of strand exchange on the b  and h strands respectively .
the 2-ap substituted junctions were titrated with ctgen1 in the presence of 1  mm ca ions to prevent cleavage .
2-aminopurine 3 nt distant from the junction exhibited no significant change in fluorescent intensity on addition of ctgen1 .
by contrast , the two 2-ap bases located immediately adjacent to the junction exhibited marked increases in intensity ( figures 7b and 7c ) .
the 2-ap 5 to the point of strand exchange on the r strand increased to a plateau level reflecting an 40-fold enhancement of fluorescence , while that 3 to the junction increased 10-fold .
an increase of such magnitude indicates a major opening of the junction , probably involving local loss of base - pairing , although clearly this effect does not extend as far as the third base pair .
we have previously observed a similar level of disruption of a junction by the yeast mitochondrial enzyme cce1 ( dclais and lilley , 2000 ) .
we repeated the same analysis on equivalent dna species constructed to represent the product of junction resolution .
although 2-ap located at the r-1 position exhibited an increase in fluorescence intensity on addition of ctgen1 , this was only half that observed for the complete junction ( figure  7c ) .
this was confirmed by activating the ctgen1 in the complex with the junctions by addition of an excess of mg ions , whereupon the 2-ap fluorescence intensity reduced by 50% ( figure  s7 ) .
its protein architecture is closely related to those of the other fen1-xpg family members , including its active site , which is that of a standard two - metal - ion - mechanism nuclease ( figure  4 ) .
a flap can be considered to be half of a four - way junction , and so it might be anticipated that dimerization of a fen - type domain could generate a junction - resolving enzyme .
our structural studies show that ctgen1 is effectively an elaborated form of fen1 that has substantially dispensed with the helical arch that is not required for the selection of a single - stranded section . instead , this region of ctgen1
is probably involved in recognition of the central structure of the junction , and in part contributes to the dimerization domain , while a new c - terminal section makes additional contacts to the dna so as to increase the affinity and selectivity for the structure of the junction once dimerization has occurred . in our experiments
, we crystallized a wild - type sequence in the  presence of mg ions . under these conditions ,
ctgen1 is fully active , and the species crystallized is the product of resolution cleavage . the crystallization process has selected a single product of enzymatic cleavage , containing the 30 nt r strand from the junction and the 3 half of the h strand and the 5 half of the x  strand , i.e. , the product with r and x arms .
the expected one nucleotide 3 overhang on the x strand is not visible in the electron density , presumably because it is too mobile .
similarly , the 5 nucleotide of the x strand is unpaired and not visible in the electron density .
although the functional unit observed in the crystal corresponds to one of the products of resolution ( figure  1 ) , protein - protein interaction between the ctgen1 monomers in the crystal lattice generates a structure that is clearly related to that of the dimeric enzyme bound to a four - way junction ( figure  5 ) .
the two dna species are held by the proteins so that the uncleaved arms ( these would be the h and x arms of the complete junction ) are close to coaxial , while cleaved arms ( b and r in the junction ) are mutually perpendicular and perpendicular to the h - x axis .
this is exactly the disposition of arms that was deduced for the junction in solution from the comparative gel electrophoresis experiments ( figure  6 ) .
we found that it was possible to reconnect the dna strands in the complex observed in the crystal to generate an intact four - way junction without altering the disposition of the arms ( figure  5e ) , but this required a degree of helical opening at the junction center .
this is fully in agreement with the observation of enhanced chemical reactivity and 2-aminopurine fluorescence on addition of ctgen1 to a four - way junction ( figure  7 ) .
the opening of the four - way junction by ctgen1 is similar to that induced by the majority of junction - resolving enzymes ( dclais and lilley , 2008 ) .
the dimerization interface observed in the crystallized complex primarily comprises the helices 4 , 5 , and 14 and associated loops ( figures 5c and 5d ) .
dimerization involves a relatively small contact area of 530   , consistent with a low tendency of the protein to dimerize in free solution ( freeman et  al . , 2014 ) .
binding to a dna junction is strongly cooperative , with a hill coefficient > 3 ( table s1 ) .
these observations suggest that ctgen1 exists in solution primarily in monomeric form and dimerizes on binding to the junction .
this provides an opportunity for regulation of activity that is not possible in resolving enzymes from lower organisms that exist in solution in dimeric form .
binding of active monomeric ctgen1 to a junction could potentially generate undesirable unilateral cleavage of a four - way junction prior to dimer formation .
we postulate that monomeric ctgen1 activity is suppressed by a partially disordered active site , providing a failsafe mechanism .
the loop between 3 and 5 where the helical arch in fen1 is located is disordered in the product complex , yet contains a number of basic residues that are likely to play a role in both branch point distortion and the cleavage reaction .
the disordered region is in close proximity both to the dimer interface and to the reconnected strands in our model of the intact four - way junction , suggesting that this part of the active site only becomes structured when a ctgen1 dimer is bound to  the intact junction , thereby activating the enzyme for cleavage .
according to this hypothesis , ctgen1 monomer bound to a four - way junction would be relatively inactive , thus minimizing unilateral cleavage of four - way junctions and preventing more promiscuous activity on flaps and other kinds of junctions .
the structure of the dna product bound to ctgen1 , and the  reduction in 2-aminopurine fluorescence on cleavage by ctgen1 , indicates that the dna structure at the center of the intact junction is more open than the product , where the base adjacent to the cleavage site is paired .
moreover , the position of the 5-terminal phosphate relative to the two metal ions in the cleavage site in the product complex is not suitable for in - line attack by a metal - bound water molecule , so the geometry in the active site must have rearranged after cleavage .
it is also very probable that the metal ions have moved from their positions prior to the cleavage reaction .
this relaxation is in contrast with what was observed in the post - cleavage complexes of fen1 and exo1 , where the scissile phosphate remains bound to the metal ions , and the adjacent base is unpaired .
this could explain our observation that in the resolution of a junction , the second cleavage reaction occurs ten times faster than the first ( freeman et  al .
if relaxation of the dna structure following first strand cleavage leads to a readjustment of the structure of the complex so that the second strand is better accommodated into the active site , this could lead to an acceleration of the hydrolytic reaction .
this would increase the probability that bilateral cleavage will occur during the lifetime of the enzyme - junction complex and thus ensure a productive resolution of the junction , with a lower probability of release of a semi - resolved junction .
the structure presented here reveals how ctgen1 is specific  for the structure of a four - way dna junction and suggests how it ensures that a productive resolution results from the interaction .
full experimental details are presented in the supplemental information . c.  thermophilum gen1 1 - 487 with a c - terminal six - histidine tag was expressed in e.  coli bl21(de3 ) ril ( stratagene ) .
ctgen1 was purified by ni - nta affinity , heparin , gel filtration , and ion exchange chromatography .
purified ctgen1 migrated as a single band on an overloaded polyacrylamide gel in the presence of sds .
purified ctgen1 was mixed with a four - way dna junction based on junction 3 ( duckett et  al . , 1988 ) and comprising 15  bp in each helical arm ( 100  m each of ctgen1 monomer and dna junction ) .
equal volumes of dna and protein were mixed in a final concentration of 100  mm hepes ( ph 7.5 ) , 2  mm mgcl2 , 20% peg10000 , and incubated with the same buffer using hanging drop vapor diffusion at 7c .
crystals were soaked in cryo - protectant , dehydrated by vapor diffusion equilibration , and stored under liquid nitrogen .
initial phases were acquired from the sad data by locating the eight selenium atoms with autosol in the phenix suite ( adams et  al . , 2010 ) .
the initial model was generated automatically by phenix autobuild wizard and then applied to the native datasets by molecular replacement using phaser ( mccoy et  al . , 2007 ) .
datasets for the complexes with native ctgen1 in mg and mn and the se - methionine - substituted ctgen1 were obtained using synchrotron x - radiation at a resolution of 2.5 , 2.6 , and 3.15   , respectively .
rates of cleavage of junction 3 by wild - type and mutant ctgen1 were measured under single - turnover conditions .
the fraction of dna cleaved at time t ( ft ) was fitted to:(equation 1)ft = ff(iexp(kct ) ) .
data were analyzed as fraction dna bound ( fb ) versus protein concentration and fitted to:(equation 2)fb=1/(1+(kd / pt)n),where kd is the dissociation constant , pt is the total protein monomer concentration .
the six possible dna junctions with two 40  bp and two 14  bp arms were incubated with 100  nm ctgen1 , loaded onto a polyacrylamide gel in the presence of 2  mm cacl2 , and run under native conditions .
junction 3 with and without ctgen1 was reacted with 1  mm kmno4 for 2  min at 25c and site - specifically cleaved by incubation with 1  m piperidine at 95c for 30  min .
junction 3 and its corresponding r - strand resolution cleavage product were prepared with adenine nucleotides substituted by 2-aminopurine at selected single positions .
steady - state fluorescence emission spectra were recorded between 330  nm and 460  nm in 1  nm intervals with excitation at 315  nm .
 summarywe present the crystal structure of the junction - resolving enzyme gen1 bound to dna at 2.5   resolution . 
 the structure of the gen1 protein reveals it to have an elaborated fen - xpg family fold that is modified for its role in four - way junction resolution . the functional unit in the crystal is a monomer of active gen1 bound to the product of resolution cleavage , with an extensive dna binding interface for both helical arms . within the crystal lattice , 
 a gen1 dimer interface juxtaposes two products , whereby they can be reconnected into a four - way junction , the structure of which agrees with that determined in solution .  
 the reconnection requires some opening of the dna structure at the center , in agreement with permanganate probing and 2-aminopurine fluorescence . 
 the structure shows that a relaxation of the  dna structure accompanies cleavage , suggesting how second - strand cleavage is accelerated to ensure productive resolution of the junction . 
Introduction
Results
Discussion
Experimental Procedures