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THEJOURNALOF BIOLOGICAL CHEMISTRY Vol. 262,No. 19,Issue of July 5,pp.

9160-9165, 1987
0 1987 by The American Society of Biological Chemists, Inc. Printed in U.S.A.

Construction of a Synthetic Holliday Junction Analog and


Characterization of Its Interaction with a Saccharomyces cereuisiae
Endonuclease ThatCleaves Holliday Junctions*
(Received for publication, August 12, 1986)

David H. Evans$ and Richard Kolodnerg


From the D a m Farber Cancer Institute, Boston, Massachusetts 02115 and the Department of Biological Chemistry, Haruard
Medical School, Boston, Massachusetts 02115

W e describe the construction and characterization of bacteriophage T 4 gene49 encodes endonuclease VI1 (12). This
an oligonucleotide Holliday junction analog and char- endonuclease appears to be required for packaging T 4 DNA
acterize its interaction with a Saccharomyces cerevis- intophageheadsand catalyzes the cleavage of multiply
iae endonuclease that cleaves Holliday junctions. A branched T4 replication intermediates and other structures
Holliday junction analog containing four duplex arms containing Holliday junctions. The bacteriophage T 7 gene3
and 54 base pairs was constructed by annealing four product is required for both recombination and for the pro-
unique synthetic oligonucleotides. Mixing curve anal- duction of DNA precursors for DNA synthesis (13). The T7
ysis showed that the complex contained a 1:l: 1:l mol
ratio of the four unique sequence strands. In addition, gene3 encodes the T7 endonuclease I (14) which is a single-
a linear duplex with a sequence identical to two of the stranded DNA-specific endonuclease that also cleaves Holli-
junction arms was also constructed for use as a control day junctions (13). Recently, Saccharomycescereuisiae has
fragment. High resolution gel exclusion chromatogra- been shown to contain a Holliday junction specific endonu-
phy was used to purify and characterize the synthetic clease, although this enzyme has not yet been implicated in
junction. The synthetic Holliday junction was found to any particular aspectof nucleic acid metabolism(15,16). The
be a specific inhibitor of a S. cerevisiae enzyme that bacteriophage X int protein is required for integrative recom-
catalyzes the cleavage of Holliday junctions. Under bination and has been shown to resolve Holliday junctions
standard cleavage conditions, 50%inhibition was ob- (5). However, the X int protein appears to differ from the
served at a synthetic Holliday junction to substrate former three enzymes in thatit only resolves Holliday junc-
ratio of 711, whereas no inhibition by linear duplex tions constructedfrom att sites and the products of resolution
was observed at molar ratios in excess of 150/1. Kinetic do not contain any broken phosphodiester bonds.
analysis showed that Holliday junction was a compet- Most studies characterizing enzymes that cleave Holliday
itive inhibitor of the reaction and had an apparent K i junctions have focused on determining the position of the
= 2.5 nM, although the mode of inhibition was complex. cleavage sites relative to the Holliday junction. The most
The synthetic Holliday junction was not asubstrate for extensively used substrates for these studies have been cova-
the enzyme, but was found to form a specific complex
lently closed circular DNAs containing palindromes. When
with the enzyme as evidenced by polyacrylamide gel
electrophoresis DNA binding assays. heated, suchmolecules will extrude a cruciform structure that
contains a Holliday junction at its base (17) and provides a
convenient substrate for the enzymes described above. Using
these types of substrates, T4 endonuclease VI1 and the T7
endonuclease I have been shown to cleave cruciform contain-
DNA structures in which two duplex DNA molecules are ing DNAs at sites symmetricallylocated near the baseof the
joined to each other by a reciprocalsingle-stranded cross-over Holliday junction (12, 13). The products formed were linear
have been postulated to be intermediatesin a number of
duplex molecules with hairpin ends
containing single-
cellularprocesses. Replication (l),telomere resolution (2),
stranded breaks thatcould be sealed with DNA ligase. The s.
and general (3, 4) and site-specific recombination (5) are all
cereuisiae endonuclease is also thought to cleave cruciform
processes that may generate such structures now commonly
containing DNA ina similar fashion except that thisenzyme
referred to as Holliday junctions (6). Their importance has
prompted physical searches for such structures and the iso- has not beenas extensively studied as the T4 andenzymes T7
lation of molecules containing Holliday junctions has been (15, 16). The exact distance between the cleavage sites and
described in a number of systems (7-11). the Holliday junction remains somewhat uncertain because it
The processing of Holliday junctions could be catalyzed by is unclear if the boundenzyme alters the extentof cruciform
specific endonucleases and several endonucleases that cleave extrusion and because in the case of the T4 endonuclease
Holliday junctions have been described. The Escherichia coli there may be preferred sequences that are cleaved (12).
Substrates lacking the sequence symmetry present in nat-
* This work wassupported by National Institutes of Health Grant ural Holliday junctions and cruciformsprovide a way by which
GM29383 (to R. K.). The costs of publication of this article were the problem of junction migration can becircumvented. Sub-
defrayed in part by the payment of page charges. This article must strates capable of only very limited branch migration have
therefore be hereby marked “aduertisement” in accordance with 18 been constructed by annealing phage X att sites (8) or M13
U.S.C. Section 1734 solely to indicate this fact. single strands (18).Kallenbach et al. (19) have constructed
$ Postdoctoral Fellow of the Alberta Heritage Foundation for Med-
ical Research. four-stranded junctions from synthetic oligonucleotides and
§ T o whom correspondence should be addressed Dana Farber characterized them extensively by NMR techniques (20). In
Cancer Inst., 44 Binney St., Boston, MA 02115. order to prevent any exchangeof base pairing, however, the

9 160
Synthetic Holliday Junction Analog 9161
junctions constructed by these latter authors lack the four were electrophoresed through 0.8% agarose gels in 40 mM Tris, 5 mM
symmetricalbasesexpectedto be found at the core of a sodium acetate, 1 mM EDTA, pH 7.9, buffer containing 0.5 pg/ml
ethidium bromide. Parallel lanes containing standard amounts of
Holliday junction. We describe here the construction of a linear pBR322::PAL114 or pBR322 DNAs were also run. Gels were
somewhat larger synthetic Holliday junction that contains photographed with Polaroid Type 665 positive/negative film and
these four symmetricalbasesandillustrate how suchan DNA quantitated by densitometry. One unit of enzyme cleaves 1 ng
analog can be used t o analyze the substrate specificity of an PAL114 DNA in 1 h at 30 "C. This is 0.34-fmol cruciform junctions
endonuclease thatrecognizes Holliday junctions. given a molecular weight of 2.96 x lo6 g/mol for pBR322::PAL114
(22).
MATERIALS ANDMETHODS Cleavage of Oligonucleotide and Sequencing Reactions-Oligonucle-
otides were 5'-end-labeled in 50-pl reactions containing 50 mM Tris-
Synthesis of Oligonucleotides-Oligonucleotides were synthesized HCl, pH 7.5, 10 mM MgCl,, 2 mM dithiothreitol, 0.1 mM spermidine,
on an Applied Biosystems Model 380A DNA synthesizer using phos- 0.1 mM EDTA, 200 pmol of [Y-~'P]ATP(290,000 dpm/pmol), 100
phoramidities and other chemicals purchased from the manufacturer. pmol of oligonucleotide,and 3.5 units T4 polynucleotide kinase. After
Crude yields were typically 100-200 OD254 units of each strand with 30 min at 37 "C, 2 pl of 100 p~ unlabeled ATP was added and the
lengths of 25-34 nucleotides. Molar extinction coefficients were cal- reaction continued 30 min. Reactions were stopped by the addition
culated from the base composition to be (see Fig. l): strand l,e254 = of 0.7 pmol of ATP and 1pmol of EDTA in 4 pl, additional required
216,000; strand 2, e254 = 216,000; strand 3, e254 = 227,000; strand 4, oligonucleotides were added, the solution was heated 5 min at 65 "C,
f254 = 195,000; strand 5, e254 = 268,000; strand 6, c2% = 268,000. and after cooling the labeled oligonucleotide complex wasisolated by
Purification of Oligonucleotides-Detritylated oligonucleotides ob- HPLC. Specific activities were adjusted to 60,000 cpm/pmol with
tained from the synthesizer were deprotected by incubating -1 ml of unlabeled material.
oligonucleotide with 3 ml 58% ammonium hydroxide overnight at Radiolabeled oligonucleotides were digested under standard con-
55 "C. The oligonucleotides were then taken todryness in a vacuum ditions in 100-pl reactions containing 140 units of s. cerevisiae
centrifuge and resuspended with H,O. Samples containing 30 OD,,, endonuclease and 500 fmol of labeled oligonucleotide substrate. To
units of oligonucleotide were denatured by heating one part oligonu- inhibit a trace of exonuclease (present at a level that removes -0.2
cleotide with three parts probe buffer (99% formamide, 11mM NaOH, nucleotide/end/h under these conditions) 5 pmol of unlabeled linear
0.05% xylene cyanol, 0.05% bromphenol blue) at 100 "C for 1 min duplex was added to all reactions. The linear duplex was observed
and then fractionated by electrophoresis through a 20% acrylamide (see below) to have no effect on the endonuclease activity. Aliquots
gel containing 50% (w/v) urea, 0.09 M Tris borate, pH 8.3, 2.5 mM (50 p1) were removed at time 0 and after 2 h at 30 "C and the DNAs
EDTA. Full-length products were identified by UV shadowing, eluted purified by Sep-Pak chromatography in the presence of 50 pgof
from the gel, and purified on WatersC-18 Sep-Pak columns according carrier tRNA (23). The lyophilized products were redissolved in 10 pl
to the method of Lo et al. (23). Samples were resuspended at 200 p M of probe buffer and fractionated by electrophoresis through 0.3-mm
(oligonucleotide) in 10 mM Tris, pH 8.0, 1 mM EDTA, and stored thick 20% acrylamide/urea gels as described above.
frozen at -20 "C. Yields were typically 30-50% of material applied to Sequence markers were prepared from appropriate 5'-32P-labeled
the gels. oligonucleotides as described (24) except that reaction times were
Hybridization of Strands-Strands were mixed in appropriate ra- adjusted as necessary to give good product distributions. Autoradiog-
tios at strand concentrations of 5-80 p~ and 5 M NaCl added to 0.1 raphy was performed at -70 "C with Kodak X-Omat AR film and
M. The solutions were then heated at 65 'C for 5 min and then two fluorescent intensifying screens (Du Pont).
incubated a t room temperature. Hybridization appeared to be com- Polyacrylamide Ekctrophoretic DNA Binding Assays-Standard
plete within 5 min of returning to room temperature. In some exper- reactions (10 pl) contained 50 mM Tris-HC1, pH 7.8, 40 mM NaCI, 1
iments 5 mM MgC12 was included but this was later found to be mM dithiothreitol, 0.1 mg/ml acetylated bovine serum albumin, 10
unnecessary. fmol of 32P-end-labeledpolynucleotide complex (-3000 cpm), soni-
High Performance Liquid Chromatography-A Waters HPLC'sys- cated E. coli DNA (a gift of Dr. M.A. Osley, Dana Farber Cancer
tem consisting of automatic gradient controller, Model 441 absorb- Institute, Boston, MA, -500-bp in length) and 0.4 units (8 ng) of
ance detector operating at 254 nm, Model 510 pumps, Model U6K double-strand DNA cellulose purified S. cereuisine endonuclease.
liquid injector, and Protein-Pak 300 SW size-exclusion column was After adding enzyme, reactions were incubated 20 min a t 20 "C and
used in allchromatography experiments. Column buffer (10 mM Tris- then 2 p1 of50% (w/v) glycerol, 5 mM Tris-HCI, pH 8.0, 0.5 mM
HCl, 100 mM NaCl, 1 mM EDTA, pH 7.5) was filtered through type EDTA, 0.05% bromphenol blue added, and the samples electropho-
GSTF 0.22-pm filters (Waters Associates, Milford, MA) before use. resed 3 h at 4 "C at 200 V. Gels contained 8% acrylamide (145:5
To control the temperature the column, precolumn, and inlet tubing acrylamide:bisacrylamide),1mM EDTA, 3.3 mM sodium acetate, and
were immersed in a regulated water bath. 6.7 mM Tris, pH 7.5, and were run in the same buffer.
Enzymes-S. cerevisine endonuclease was purified essentially as
described by Symington and Kolodner (15) with the following modi- RESULTS
fications. Strain LBLl/n was grown on arotaryshaker at room
temperature and pretreated 5 h with 0.01% (v/v) methyl methane Designing a Synthetic Holliday Junction-Fig. 1 illustrates
sulfonate (Kodak). Methyl methanesulfonate was added when cells the two oligonucleotide complexesthat we have prepared. The
reached Am = 1 and cells harvested 5 h later at Am = 2-3 (late log). Holliday junction makesuse of two synthetic oligonucleotides
Most experiments described here used enzyme purified through the that were originally prepared for other studies (strands 1and
single-strand DNA cellulose step as described (15). Specific activity
of the enzyme preparation (see below) was 39,000 units/mg and was
2 (25)) and two that were specifically designed to generate a
approximately 300-fold purified relative tothe crude lysate. The uniquebase-pairingmrangement(strands 3 and 4). This
enzyme was further purified to a specific activity of -50,000 units/ structure is potentially capable of base pairing right to the
mg by chromatography on double-strand DNA cellulose for oligonu- junction as are true Holliday junctions. The base pairs sur-
cleotide cleavage experiments. T4 polynucleotide kinase was purified rounding the junctionwere chosen to prevent branchmigra-
as described by Panet et al. (21). tion into alternate base-pairing arrangements and the arm
Enzyme Assays-S.cerevisiae endonuclease was assayed for its
ability to cleave pallindromes extruded in plasmid pBR322::PAL114 lengths are all different. Different arm lengths were used to
(22). Standard assays (100 pl) contained 10pg/ml DNA, 8 mM MgC12, prevent uniformly distributed exonuclease action from ap-
50 mM Tris-HC1, pH 7.8,O.l mg/ml acetylated bovine serum albumin, pearing as symmetrical endonuclease incisions in cleavage
10 m M dithiothreitol, and enzyme. After incubation a t 30 "C for 1 h, experiments. It should be noted that unlike true Holliday
the DNA waspurified by phenol extraction, ethanolprecipitated, and junctionsthe sequences of the four armspresentinthis
recut with P o d 1 in 15-pl reactions containing 6.7 mM Tris-HC1, pH structure are all different. In addition, a34-bplong linear
8.0, 60 mM NaCl, 6 mM MgCl,, 6 mM (3-mercaptoethanol, 0.1 mg/ml
acetylated bovine serum albumin, 0.67 mM EDTA, and 5 units PvuII duplex was synthesized as a control. The sequence is that of
(New England Biolabs, Beverly, MA). After 1 h a t 37 "C, samples the vertical arms of the Holliday junction (Fig. 1).
Construction of Oligonucleotide Complexes-HPLC sizing
The abbreviations used are: HPLC, high pressure liquid chro- columns are capable of resolving single strands from duplex
matography; bp, base pair. andfour-stranded complexes. The limits of resolution on
9162 Synthetic Holliday Junction Analog
5
GC
GC 0.
CG
CG
TA
AT
GC
AT
TA
CG
TA
5 CG 6
GC
AT
GC
CG
GC P
AT
GC
CG
TA
CG
GC
CG
GC
TA
AT
CG
CG
AT
AT AT
GC GC 08 10 I2 14
CG CG Strand role ratio Half-junction mole ratio
TA TA
Y Y FIG.2. A, strands 2 and 3 anneal 1:l to form half of the Holliday
junction. Strand 2 (150 pmol) was mixed with100-200 pmol of strand
3 in a total volume of 25-35 pl and hybridized as described under
FIG.1. Structure of synthetic Holliday junction and control “Materials and Methods.” Samples were then chromatographed and
duplex. Single strands range in size from 25 to 34 bases in length. the relative amountsof dimer and monomer quantitated using peak
The sequence of the linear duplex is exactly the same as the long elution heights. Breaks inthe curves at 1:l mol ratio are observed as
vertical arm of the Holliday junction. Although drawn as a planar expected, demonstrating that the dimer contains strands 2 and 3 in
structure the Holliday junction is probably tetrahedral in solution equimolar quantities. E , formation of Holliday junctions from two
(18). half-junctions.Duplexes containing equimolar mixesof strands (1 +
4) or (2 + 3) were purified by HPLC and quantitated by UV spec-
troscopy. Strands (2 + 3) (39-pmol complex)were mixed with 28-55
these columns are illustratedby the observation that 34-mers pmol of strands (2 + 3) in 40-60 pl, hybridized,and chromatographed
are fairly well separated from 25-mers. This technology can as described above. Breaksat 1:l mol ratio confirmthat the synthetic
be exploited to quantitate complex concentrations and dem- junctioncontains strends 1, 2, 3, and 4 inequimolarquantities.
onstrate that appropriate strands form two- and four-stranded Monomers, (-O-); half-junctions (*-); and Hollidayjunction,
complexes with the expected strand stoichiometry. (-X-).

In order to demonstrate that the oligonucleotides can form


a four-stranded Holliday junction, pairs of strands were first ,Tetramer
annealed in varying ratios. By fixing the amountof one strand
and varying the amount of the second one would expect to
see the amount of half-junction increases until the second
strand is in excess. Then, the amount of half-junction becomes
limited by the amount of the first strand and remains con-
stant. Conversely, the total amount of monomer would be
expected to decrease as half-junction is formed then increases
again as the second strand becomes present inexcess. Fig. 2A
illustrates such a mixing experiment. As predicted, strands 2
and 3 anneal 1:l and similarly 1:l hybridization of strand 1
with strand 4 and strand 5 with strand 6 was also observed
(not shown). I I I I I I I I
Formation of a four-stranded complex can be demonstrated
in the same way, except 1:1 mixtures of half-junctions were 0 2 4 6 8 1 0 1 2 1 4
mixed instead of single strands. Within the limits of error
Elution Volume (mL)
(extinction coefficients are not known with absolute accu-
racy), a four-stranded complex consisting of oneeach of FIG.3. HPLC analysis of a synthetic Holliday junction.
strands 1, 2, 3, and 4 was observed (Fig. 2 B ) . Buffer temperature was controlled by warming the column and 30
cm of inlet tubing ina regulated water bath. Samples (16 pmol in 50
Stability of the Holliday Junction-Rechromatography of pl) of column-purified Holliday junctionwere rechromatographed at
purified Holliday junction does not give a single peak, but 30 “C. Half-junctions andfree monomers that form as the junctions
rather free dimer and monomer components reappear (Fig. disassociate can be seen; the relative proportions of tetramer, dimer,
3). This is also apparent in Fig. 2 where at 1:l mol ratios of and monomerspeciesprovides a qualitative measure of junction
component strands not all of the components form higher stability under these conditions.
molecular complexes. This is due to chromatographic sepa-
ration of the strands that are in dynamic equilibrium with shown) and under the reaction conditions described here the
one another. The equilibrium distribution of the strand can junction exists primarily in a four-stranded configuration.
be calculatedfrom the peak heights and from these values the Size Behauior-The unusual structureof the four-stranded
free energy can be estimated. The resultsof this analysiswill complex affects its mobility during electrophoresis through
be published elsewhere: but as can be seenby inspection of agarose and acrylamide gels (data not shown). In order to
Fig. 3, this junction is stable at moderate temperatures and minimize disassociation into component strands 15% acryl-
salt concentrations. This stability is enhanced by MgC12 (not amide gels were run at 4 “C in the presence of0.5 pg/ml
ethidium bromide. Under these conditions theHolliday junc-
* D. H. Evans and R. Kolodner, manuscript in preparation. tion (which contains 54 bp) migrated as if it were a 172-bp
Synthetic Holliday Junction Analog 9163
linear DNA. Under other conditions (agarose gels at room polyacrylamide gel electrophoretic DNA binding assays (26).
temperature with or without ethidiumbromide), the Holliday Under the conditions of the binding assay, the synthetic
junction migrated as would linear fragments ranging in size Holliday junction exists in equilibrium with the denatured
from 91 to 230 bp. We have not studied the electrophoretic forms discussed above (Fig. 5, lane 1). Addition of the enzyme
behavior of this molecule in detail; however, our resultsclearly preparation resulted in the formation of several species that
show that, asexpected, the four-stranded complex has unusual migrated more slowly than the unbound Holliday junction
hydrodynamic properties. Similar behavior has been described (Fig. 5, Zane 2 ) . When increasing amounts of nonspecific
by others (18, 19). competitor E. coli DNA was added to the binding reactions
Interaction with a Saccharomyces cereuisiae HollidayJunc- along with the synthetic Holliday junction, the slowly migrat-
tion Specific Endonuclease-If the synthetic Holliday junction ing species were gradually competed away (Fig. 5, lanes 3-
hasastructure analogous to naturally occurring Holliday IO). All except one protein-DNA complexwere competed
junctions it should interact with enzymes that recognize such away by the addition of 22 ng of competitor DNA, and the
junctions. Initially, we have studied the cleavage of the syn- most stable complex was still formed in the presence of 220
thetic Holliday junction by the S. cereuisiae Holliday junction ng of competitor DNA. In control experiments in which
specific endonuclease. 5’-32P-labeled Holliday junction was binding reactions were carried out with linear duplex control
digested with S. cereuisiue endonuclease, and the reaction DNA, all of the protein-DNA complexes formed were com-
products were analyzed by electrophoresis through acrylamide peted away bythe addition of 22 ng of competitor DNA. This
gels. In anumber of experiments (data notshown) no cleavage suggests that the most stable complex formed with the syn-
products were detected. This indicated that the synthetic thetic Holliday junction represents specific binding to the
Holliday junction wascleaved at (5% of the rate of the Holliday junction.
standard pBR322::PAL114 substrate and was therefore un-
likely to be a substrate for the enzyme. DISCUSSION
We have studied the ability of the synthetic Holliday junc- Design and Construction of a SyntheticHolliday Jumtion-
tion to inhibit the cruciform cleavage reaction catalyzed by Natural Holliday junctions that join duplex DNA molecules
the S. cerevisiae endonuclease. As illustrated in Fig. 4A, the at regions of homology can move by branch migration due to
synthetic Holliday junction is a specific inhibitor of this the presence of sequence symmetry surrounding the joint.
reaction. The linear duplex has no effect on the S. cereuisiue This feature complicates the ability to isolate naturally oc-
enzyme even at concentrations in excess of 100 nM, whereas curring Holliday junctions and study their structure because
only moderate concentrations of synthetic Holliday junction such structures are heterogeneous and sometimes unstable.
cause significant inhibition. The inhibition appears to be The ability to branch migrate also complicates the study of
competitive and from the substrate concentration (3.4 nM) enzymes that cleave Holliday junctions because the junction
and K,,, (Fig. 4B, 2.3 nM) a K j of 2.5 nM under these conditions can exist in many positions relative to the sequence of the
can be calculated. The mode of inhibition is complex and substrate. In addition, it is also possible that interaction with
further kinetic analysis (Fig. 4B) indicated that the enzyme such enzymes can change the position of the Holliday junc-
displayed substrate inhibition at substrate concentrations in tion. Kallenbach et al. (19) have described the use of synthetic
excess of 4 nM and that the synthetic Holliday junction was oligonucleotides to construct Holliday junction analogs that
a markedly better inhibitor at low substrate concentrations contain fixed junctions. In this communication we describe
than athigh substrate concentrations. the construction of a synthetic Holliday junction analog for
The binding of 5’-32P-labeledsynthetic Holliday junctions use in studying enzymes that interact with Holliday junctions.
tothe S. cerevisiae endonuclease has been studied using The Holliday junction analog described here differs from

T
i
A. B.

140 t

/ I ,, I
-x) 0 10 x) 100 -10 -a5 o 05 IO E 20 25

CII (nM) ’/lcrc!esl (nM-l)


FIG. 4. A , Dixon plot illustrating specific inhibition of S. cereuisiue endonuclease by the synthetic Holliday
junction. Standard reactions (100 pl) containing 57 units of S. cereuisiue endonuclease and 0.34 pmol of PAL114
DNA were supplemented with oligonucleotide complexes as indicated and enzyme activity quantitated as described
under “Materials and Methods.” Under these conditions a Kc of 2.5 nM can be calculated (see text). Holliday
junction, (0);linear duplex, (0).B , Lineweaver-Burk analysis of the inhibition of S. cereuisioe endonuclease by a
synthetic Holliday junction. Reactions (100 pl) containing 57 units of S. cereuisiue endonuclease were prepared as
described except that plasmid concentrations were varied as illustrated and the initial rate of plasmid cutting
measured in the presence or absence of 5 nM synthetic Holliday junction. Under these conditions K , and V , are
2.3 nM (eireks) and 56 fmol/h, respectively. No addition, (0);plus 5 nM synthetic Holliday junction, (0).
9164 Synthetic Holliday Junction Analog
1 2 3 4 5 67 8 910 1112 13141516171819 x) thermore, a t low substrate concentrations enhanced inhibi-
tion is also observed. This could reflect a complex, possibly
,--Holliday junction7 r Linear duplex-i cooperative, mode of interaction between the enzyme and its
substrate. Another possible explanation for these kinetics is
I Enzyme I
- + + + + + + + + + - + + + + + + + + + that theyreflect a binding competitionwhich normally favors
plasmid binding even if the actual affinities for plasmid and
Competitor DNA (ng/reaction)I- synthetic junctions arevery similar. This isbecause nonspe-
cific binding and facilitateddiffusion can preferentially target
enzyme molecules to plasmid junctions much as EcoRI pref-
erentially associates with recognition sites inbedded in large
pieces of DNA (27). This competionwill, however, be critically
dependent upon the relative concentration of the two types
of binding sites and under conditions where binding to the
oligonucleotide is favored ([SI < K,,, or [I] >> [SI) one would
expect enhanced inhibition like that observed. This type of
behavior could also be described as very slow, tight binding
inhibition (28). Moredetailedkineticanalysis,additional
information on the structureof the enzyme, and footprinting
experiments are clearly required before this question can be
resolved.
The syntheticHolliday junction was not a substrate for the
S. cereuisiae Holliday junction specific endonuclease. There
are several possible explanations for this observation. First,
the junction may be too small. Thus, the enzyme may not
bind it sufficientlywell for it to serve asa substrate. A point
that may argue against this again comes from observationsof
EcoRI. EcoRI binds with similar specific activity to theEcoRI
recognition site in 34- or 4363-bp DNAs implying flanking
FIG. 5. Specific binding of S. cerevisiae endonuclease to nonspecific sequences and charges on oligonucleotides of this
synthetic Holliday junction. Oligonucleotides were 5’-3ZP-end- size provide few, if any, additional binding sites (29). Another
labeled andincubatedwithorwithout 0.4 units of S. cereuisiae affect of size is the possibility that the cleavage sites are
endonuclease in the presence of varying amounts of sonicated E. coli located more than 14 bpaway from the junction (the largest
DNA as described under “Methodsand Materials.” After incubation
samples were electrophoresed on 8% polyacrylamide gels at 4 “C, symmetrical cuttingwe could observe).This possibility cannot
dried, and autoradiographed with fluorescent intensifier screens for be ruled out based on plasmid cruciform cleavage experiments
2 days at -70 “C. Lanes 1-10, synthetic Holliday junction; lunes 11- since the junction location is not known with precision in
20, linear duplex control. S. cereuisiae endonuclease was omitted in such experiments.
lanes 1 and 11. Another possible explanation for the failure to observe
cleavage of the synthetic Holliday junction is that symmetry
those of Kallenbach et al. (19) in that each duplex arm is a elements (homology) within the arms of the junction are
different length to aid in mapping specific cleavage products. recognized by the enzyme. Assuming the S. cereuisiae enzyme
In addition, we retained a one nucleotide symmetry at the is involved in general recombination or recombinational re-
core of the junction since such symmetry mightbe expected pair, it should be nonsequence specific. However,local se-
to play a role in theoverall three-dimensional structureof the quence symmetry around the Holliday junction may still be
junction. From binding studies it is apparent that four strands important to thecleavage reaction. Nonsymmetric junctions
annealwith 1:l:l:l stoichiometrytoform a structure we would not normally be formed during genetic recombination
presume tobe base-paired as illustrated Fig. in 1.The peculiar unless the recombination substrate contained a heterology, in
size behavior is consistent with thesuggestion that the arms which case the junctionwould sometimes be half-symmetric.
of Holliday junctionsaretetrahedrallyarranged (18), but The S. cereuisiae enzyme cleaves plasmid borne cruciforms,
proof of this arrangement and the details of base pairing which we presume to be half-symmetric. This isbecause the
around the junction cannot be deduced without NMR or x- excess supercoiling present in small plasmid DNAs is nor-
ray techniques. mally sufficient tocompletely extrude small inverted repeats.
Interaction of a Synthetic Holliday Junction with a n Enzyme The cleavage sites of the S. cereuisiae (15, 16) and also the
That Cleaves Holliday Junctions-The available physical evi- T 7 gene3 endonuclease (13), appear to be located within the
dence suggests that we have constructed an analog of the core symmetrically related sequences. In contrast, T 4 endonucle-
of a Holliday junction. This structure isa competitive inhib- ase VI1 cleavage siteshave been mappedtononsequence
itor of an enzyme that cleaves such junctions(Fig. 4) and this symmetric sites (12) but whether thisaisgeneral propertyof
effect is specific to the synthetic junction. Small linear oligo- these nucleases is unclear. Assuming the small size of the
nucleotides (or ends) areclearly not inhibitory (Fig. 4A). The synthetic junction is not a problem, the data presented here
synthetic junction isa very effective inhibitor with a Kionly could be taken to suggest that sequence symmetry may, in-
slightly higher than the K,,, for plasmid borne junctionswhich deed, be important to thes. cerevisiae endonuclease.
is especially curious considering its smallsize which probably It is very difficult, however, to separate sequence effects
precludes association with the enzyme by a process of one- from symmetry effects. A third possible reason that the syn-
dimensional diffusion. thetic Holliday junction is notcleaved is because the sequence
The mode of inhibition is complex. Substrate inhibition is is one that this enzyme cannot cut. Sites of genetic recombi-
observed at high substrate concentration and this effect is nation in yeasts are known to be nonrandomly distributed
pronounced in the presence of the inhibitor (Fig. 4 B ) . Fur- (30, 31) and whether this is due to nonrandom initiation of
Synthetic Holliday Junction Analog 9165
recombination or resolution remains unknown, but it is quite 10. Potter, H., and Dressler, D. (1978) Proc. Natl. Acud. Sci. U. S. A.
possible that there are DNA sequences in which Holliday 75,3698-3702
11. Thompson, B. J., Escarmis, C., Parker, B., Slater, W. C., Doniger,
junctions either do not form (32) or if formed are poorly J., Tessman, I., and Warner, R. C. (1975) J.Mol. Biol. 91,409-
resolved. Recently we have been studying the resolution of 419
other types of Holliday junctions and find quite dramatic 12. Kemper, B., Jensch, F., Depka-Prondzynski, M. V., Fritz, H.-J.,
effects of sequence on both the directionality of S. cerevisiae Borgmeyer, U., and Mizuuchi, K. (1984) Cold Spring Harbor
endonuclease cleavage and on the rate of cleavage,' and it Symp. Quant. Biol. 49,815-825
13. deMassey, B., Studier, F. W.,Dorgai,L., Appelbaum, E., and
requires further study to determine whether the lack of se- Weisberg, R. A. (1984) Cold Spring Harbor Symp. Quant. Biol.
quence symmetry or the DNA sequence per se is responsible 49,715-726
for the inability of the S. cerevisiae endonuclease to cleave 14. Sadowski, P. (1972) Can. J. Biochem. 5 0 , 1016-1023
the synthetic junction. 15. Symington, L. S., and Kolodner, R. (1985) Proc. Natl. Acud. Sci.
These experiments show that small oligonucleotides can be U. S. A. 82, 7247-7251
used to constructoligonucleotide complexes which by physical 16. West, S. C., and Korner, A. (1985) Proc. Natl. Acud. Sci. U. S. A.
82,6445-6449
and enzymatic criteria aremodels of Holliday junctions. Ques- 17. Mizuuchi, K., Mizuuchi, M., and Gellert, M. (1982) J. Mol. Biol.
tions remain regarding what features of Holliday junctions 156,229-243
are required for the cleavage reaction that is catalyzed by the 18. Gough, G. W., and Lilley, D. M. J. (1985) Nature 3 1 3 , 154-156
class of enzymes that process Holliday junctions. By con- 19. Kallenbach, N. R., Ma, R.-I., and Seeman, N. C. (1983) Nature
structing different synthetic Holliday junctions it should be 305,829-831
possible to precisely define the features of Holliday junctions 20. Wemmer, D. E., Wand, A. J., Seeman, N. C., and Kallenbach, N.
R. (1985) Biochemistry 2 4 , 5745-5749
that are recognized by these types of enzymes. 21. Panet, A., van de Sande, J. H., Loewen, P. C., Khorana, H. G.,
Raae, A. J., Lillehaug, J. R., and Kleppe, K. (1973) Biochemistry
Acknowledgments-We would like to thank Dr. C. Muster-Nassal 12,5045-5050
for helpful advice and P. Morrison for the synthesis of oligonucleo- 22. Warren, G. J., and Green, R. L. (1985) J. Bacteriol. 161, 1103-
tides. 1111
23. Lo, K.-M., Jones, S. S., Hackett, N. R., and Khorana,H. G.
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