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Construction of A Synthetic Holliday Junction Analog
Construction of A Synthetic Holliday Junction Analog
9160-9165, 1987
0 1987 by The American Society of Biological Chemists, Inc. Printed in U.S.A.
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-).
T
i
A. B.
140 t
/ I ,, I
-x) 0 10 x) 100 -10 -a5 o 05 IO E 20 25