(Received for publication, February 6, 1997)
From the Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021
Vaccinia topoisomerase binds duplex DNA and forms
a covalent DNA-(3-phosphotyrosyl) protein adduct at the sequence
5
-CCCTT
. The enzyme reacts readily with a 36-mer CCCTT strand
(DNA-p-RNA) composed of DNA 5
and RNA 3
of the scissile bond.
However, a 36-mer composed of RNA 5
and DNA 3
of the scissile
phosphate (RNA-p-DNA) is a poor substrate for covalent adduct
formation. Vaccinia topoisomerase efficiently transfers covalently held
CCCTT-containing DNA to 5
-OH-terminated RNA acceptors; the
topoisomerase can therefore be used to tag the 5
end of RNA in
vitro.
Religation of the covalently bound CCCTT-containing DNA strand to a
5-OH-terminated DNA acceptor is efficient and rapid
(krel > 0.5 s
1), provided that
the acceptor DNA is capable of base pairing to the noncleaved DNA
strand of the topoisomerase-DNA donor complex. The rate of strand
transfer to DNA is not detectably affected by base mismatches at the 5
nucleotide of the acceptor strand. Nucleotide deletions and insertions
at the 5
end of the acceptor slow the rate of religation; the observed
hierarchy of reaction rates is as follows: +1 insertion >
1
deletion > +2 insertion
2 deletion. These findings
underscore the importance of a properly positioned 5
-OH terminus in
transesterification reaction chemistry, but they also raise the
possibility that topoisomerase may generate mutations by sealing DNA
molecules with mispaired or unpaired ends.
Vaccinia topoisomerase, a 314-amino acid eukaryotic type I enzyme,
binds and cleaves duplex DNA at a specific target sequence, 5-(T/C)CCTT
(1-3). Cleavage is a transesterification reaction in
which the Tp
N phosphodiester is attacked by Tyr-274 of the enzyme,
resulting in the formation of a DNA-(3
-phosphotyrosyl) protein adduct
(4). The covalently bound topoisomerase catalyzes a variety of DNA
strand transfer reactions. It can religate the CCCTT-containing strand
across the same bond that was originally cleaved (as occurs during the
relaxation of supercoiled DNA) or it can ligate the strand to a
heterologous acceptor DNA 5
end, thereby creating a recombinant
molecule (5-7).
Duplex DNA substrates containing a single CCCTT target site have been
used to dissect the cleavage and strand transfer steps. A
cleavage-religation equilibrium is established when topoisomerase transesterifies to DNA ligands containing 18
bp1 of duplex DNA 3
of the cleavage site
(8-11). The reaction is in equilibrium because the 5
-OH-terminated
distal segment of the scissile strand remains poised near the active
site by virtue of the fact that it is stably base paired with the
nonscissile strand. About 20% of the CCCTT-containing strand is
covalently bound at equilibrium (11). "Suicide" cleavage occurs
when the CCCTT-containing substrate contains six or fewer base pairs 3
of the scissile bond, because the short leaving strand dissociates from
the protein-DNA complex. In enzyme excess, >90% of the suicide substrate is cleaved (11).
The suicide intermediate can transfer the incised CCCTT strand to a DNA
acceptor. Intramolecular strand transfer occurs when the 5-OH end of
the noncleaved strand of the suicide intermediate attacks the 3
phosphotyrosyl bond and expels Tyr-274 as the leaving group. This
results in formation of a hairpin DNA loop (5). Intermolecular
religation occurs when the suicide intermediate is provided with an
exogenous 5
-OH-terminated acceptor strand, the sequence of which is
complementary to the single strand tail of the noncleaved strand in the
immediate vicinity of the scissile phosphate (5). In the absence of an
acceptor strand, the topoisomerase can transfer the CCCTT strand to
water, releasing a 3
-phosphate-terminated hydrolysis product, or to
glycerol, releasing a 3
-phosphoglycerol derivative (12). Although the
hydrolysis and glycerololysis reactions are much slower than religation
to a DNA acceptor strand, the extent of strand transfer to non-DNA
nucleophiles can be as high as 15-40%.
The specificity of vaccinia topoisomerase in DNA cleavage and its
versatility in strand transfer have inspired topoisomerase-based strategies for polynucleotide synthesis in which DNA oligonucleotides containing CCCTT cleavage sites serve as activated linkers for the
joining of other DNA molecules with compatible termini (13). In the
present study, we examined the ability of the vaccinia topoisomerase to
cleave and rejoin RNA-containing polynucleotides. It was shown
previously that the enzyme did not bind covalently to CCCTT-containing
molecules in which either the scissile strand or the complementary
strand was composed entirely of RNA (9). To further explore the pentose
sugar specificity of the enzyme, we have prepared synthetic
CCCTT-containing substrates in which the scissile strand is composed of
DNA- and RNA-containing halves. In this way, we show that the enzyme is
indifferent to RNA downstream of the scissile phosphate, but it does
not form the covalent complex when the region 5 of the scissile
phosphate is in RNA form. Also, we assess the contribution of base
pairing by the 5
end of the acceptor strand to the rate of the DNA
strand transfer reaction.
CCCTT-containing 36-mer oligonucleotides
containing a single internal 32P-label at the scissile
phosphate were prepared by ligating two 18-mer strands (synthetic RNA
or DNA oligonucleotides) that had been hybridized to a complementary
36-mer DNA strand. The sequence of the proximal CCCTT-containing 18-mer
strand was 5-CATATCCGTGTCGCCCTT as DNA or 5
-CAUAUCCGUGUCCCUU as RNA.
The sequence of the distal 18-mer strand was 5
-ATTCCGATAGTGACTACA as
DNA or 5
-AUUCCGAUAGUGACUACA as RNA. The distal 18-mer strand was
5
-labeled in the presence of [
-32P]ATP and T4
polynucleotide kinase and then gel-purified. The sequence of the
36-mer strand was 5
-TGTAGTCACTATCGGAATAAGGGCGACACGGATATG. The
strands were annealed in 0.2 M NaCl by heating at 65 °C
for 2 min, followed by slow cooling to room temperature. The molar ratio of the 5
-labeled distal 18-mer to the proximal 18-mer and the
36-mer strand in the hybridization mixture was 1:4:4. The singly nicked
product of the annealing reaction was sealed in vitro with
purified recombinant vaccinia virus DNA ligase (14, 15). The ligation
reaction mixtures (400 µl) contained 50 mM Tris-HCl (pH
8.0), 5 mM dithiothreitol, 10 mM
MnCl2, 1 mM ATP, 10 pmol of 5
32P-labeled nicked substrate, and 160 pmol of ligase. After
incubation for 4 h at 22 °C, the reactions were halted by the
addition of EDTA to a final concentration of 25 mM. The
samples were extracted with phenol-chloroform, and the labeled nucleic
acid was recovered from the aqueous phase by ethanol precipitation. The
36-mer duplex products were dissolved in TE buffer (10 mM
Tris-HCl, pH 8.0, 1 mM EDTA). Ligation of the labeled
18-mer distal strand to the unlabeled CCCTT-containing 18-mer strand to
form an internally labeled 36-mer product was confirmed by
electrophoresis of the reaction products through a 17% denaturing
polyacrylamide gel. The extents of ligation [36-mer/(36-mer + 18-mer)] were as follows: DNA-p-DNA, 88%; DNA-p-RNA, 67%; and
RNA-p-DNA, 66%.
Recombinant vaccinia topoisomerase was expressed in
bacteria and purified via phosphocellulose and SP5PW column
chromatography as described (16, 17). Reaction mixtures for assay of
covalent adduct formation contained (per 20 µl) 50 mM
Tris-HCl (pH 8.0), 0.2 pmol of 36-mer duplex, and 1 pmol of
topoisomerase. The reactions were initiated by adding topoisomerase and
halted by adding SDS to 1% final concentration. The samples were
analyzed by SDS-polyacrylamide gel electrophoresis. Covalent complex
formation was revealed by the transfer of radiolabeled polynucleotide
to the topoisomerase polypeptide (3). The extent of adduct formation
was quantitated by scanning the gel using a FUJIX BAS1000
phosphorimager and was expressed as the percentage of the input 5
32P-labeled 36-mer substrate that was covalently
transferred to protein.
An 18-mer
CCCTT-containing DNA oligonucleotide (5-CGTGTCGCCCTTATTCCC) was 5
end-labeled in the presence of [
-32P]ATP and T4
polynucleotide kinase, then gel-purified and hybridized to a
complementary 30-mer strand to form the 18-mer/30-mer suicide cleavage
substrate. Covalent topoisomerase-DNA complexes were formed in a
reaction mixture containing (per 20 µl) 50 mM Tris-HCl (pH 8.0), 0.5 pmol of 18-mer/30-mer DNA, and 2.5 pmol of topoisomerase. The mixture was incubated for 5 min at 37 °C. The strand transfer reaction was initiated by the addition of an 18-mer acceptor strand, 5
-ATTCCGATAGTGACTACA (either DNA or RNA), to a concentration of 25 pmol/20 µl (i.e. a 50-fold molar excess over the input DNA substrate), while simultaneously the reaction mixtures were adjusted to
0.3 M NaCl. The reactions were halted by addition of SDS
and formamide to 0.2 and 50%, respectively. The samples were
heat-denatured and then electrophoresed through a 17% polyacrylamide
gel containing 7 M urea in TBE (90 mM Tris
borate, 2.5 mM EDTA). The extent of strand transfer
(expressed as the percentage of input labeled DNA converted to a 30-mer
strand transfer product) was quantitated by scanning the wet gel with a
phosphorimager.
A
36-nucleotide run-off transcript was synthesized in vitro by
T3 RNA polymerase from a pBluescript II-SK() plasmid template that
had been linearized by digestion with endonuclease EagI. A
transcription reaction mixture (100 µl) containing 40 mM
Tris-HCl (pH 8.0), 6 mM MgCl2, 2 mM
spermidine, 10 mM NaCl, 10 mM dithiothreitol, 0.5 mM ATP, 0.5 mM CTP, 0.5 mM UTP,
6.25 µM [
32P]GTP, 5 µg of template
DNA, and 100 units of T3 RNA polymerase (Promega) was incubated for 90 min at 37 °C. The reaction was halted by adjusting the mixture to
0.1% SDS, 10 mM EDTA, and 0.5 M ammonium
acetate. The samples were extracted with phenol-chloroform and
ethanol-precipitated. The pellet was resuspended in formamide and
electrophoresed through a 12% polyacrylamide gel containing 7 M urea in TBE. The radiolabeled 36-mer RNA was localized by autoradiography of the wet gel and eluted from an excised gel slice by
soaking for 16 h at 4 °C in 0.4 ml of buffer containing 1 M ammonium acetate, 0.2% SDS, and 20 mM EDTA.
The eluate was phenol-extracted and ethanol-precipitated. The RNA was
resuspended in TE. Dephosphorylation of the RNA 5
terminus was carried
out in a reaction mixture (30 µl) containing 10 mM
Tris-HCl (pH 7.9), 50 mM NaCl, 10 mM
MgCl2, 1 mM dithiothreitol, 10 pmol of 36-mer RNA, and 30 units of calf intestine alkaline phosphatase (New England
Biolabs). After a 1-h incubation at 37 °C, the mixture was
phenol-extracted and ethanol-precipitated. The phosphatase-treated 36-mer transcript was repurified electrophoretically as described above.
Vaccinia topoisomerase does not
bind covalently to CCCTT-containing RNA duplexes, nor does it form a
covalent complex on RNA-DNA hybrid duplexes in which one of the two
strands is RNA (9). Control experiments showed that the failure to form
a covalent adduct on a CCCUU-containing RNA strand was not caused by
uracil substitution for the thymine bases in the CCCTT sequence (9). To
better understand why vaccinia topoisomerase does not form a covalent
complex with all-RNA strands, we prepared 36-bp duplex substrates in
which the scissile strand was a tandem RNA-DNA or DNA-RNA copolymer and
the noncleaved strand was all DNA (Fig. 1). These
duplexes were uniquely labeled with 32P at the scissile
phosphodiester. The substrate molecules were constructed by annealing
two 18-mer oligonucleotides (one of which had been 5
32P-labeled) to a complementary 36-mer DNA strand to form a
singly nicked duplex. The 5
-labeled 18-mer strand was then joined to the unlabeled CCCTT-strand (or CCCUU strand) in a reaction catalyzed by
vaccinia virus DNA ligase. (The properties of vaccinia ligase in
joining RNA and DNA strands will be described elsewhere.) The 36-mer
duplex products were isolated and then used as substrates for vaccinia
DNA topoisomerase. We will refer to these substrates as DNA-p-DNA,
DNA-p-RNA, and RNA-p-DNA, with the labeled phosphate being denoted
by p.
Transesterification by topoisomerase at the CCCTT site will result in
covalent binding of a 3 32P-labeled 18-mer oligonucleotide
to the enzyme. The extent of covalent complex formation on the
DNA-p-RNA substrate in 10 min was proportional to input topoisomerase;
80-85% of the 36-mer strand was transferred to the topoisomerase at
saturating enzyme (Fig. 1). The same level of topoisomerase covalently
bound less than 1% of the RNA-p-DNA 36-mer strand. Hence, the
topoisomerase tolerated RNA substitution downstream of the scissile
phosphate but was impeded from forming the covalent adduct when the
CCCTT sequence was in RNA form.
We assessed the kinetics of the covalent binding reaction at a
saturating level of topoisomerase (Fig. 2). An all-DNA
36-mer (DNA-p-DNA) was bound to an end point of 21% in 2 min (Fig.
2A). The apparent cleavage-religation equilibrium constant
(Kcl = covalent complex/noncovalent complex) was
0.26, which agrees with the values of 0.2-0.25 that were reported
previously for equilibrium cleavage of a 5 end-labeled
CCCTT-containing DNA substrate (10, 11). The DNA-p-RNA 36-mer was bound
covalently to an end point of 80% in 5 min (Fig. 2A and
other data not shown). The apparent equilibrium constant for DNA-p-RNA
(Kcl = 4) was significantly higher than that
observed for the all-DNA ligand.
The RNA-p-DNA 36-mer was transferred to the topoisomerase, albeit very slowly. After 4 h, 4% of the CCCUU-containing RNA strand was bound covalently to the enzyme (Fig. 2B). An end point was not established in this experiment. However, by comparing the initial rate of covalent adduct formation on RNA-p-DNA (0.04% of input substrate cleaved per min) to the amount adduct formed on DNA-p-DNA at the earliest time point (12% in 10 s), we estimate that RNA substitution of the CCCTT-portion of the substrate slowed the rate of covalent complex formation by about 3 orders of magnitude.
DNA Strand Transfer to an RNA AcceptorRejoining of the
cleaved strand occurs by attack of a 5-hydroxyl-terminated
polynucleotide on the 3
-phosphodiester bond between Tyr-274 and the
CCCTT site. This transesterification step can be studied independent of
strand cleavage by assaying the ability of a preformed
topoisomerase-DNA complex to religate the covalently held strand to a
heterologous acceptor strand (5, 11). To form the covalent
topoisomerase-DNA donor complex, the enzyme was initially incubated
with a suicide substrate consisting of a 5
32P-labeled
18-mer scissile strand (CGTGTCGCCCTTATTCCC) hybridized to a
30-mer strand. Cleavage of this DNA by topoisomerase is accompanied by
dissociation of the 6-nucleotide leaving group, ATTCCC. With no readily
available acceptor for religation, the enzyme is essentially trapped on
the DNA as a suicide intermediate (Fig. 3). In a 5-min reaction in enzyme excess, >90% of the 5
32P-labeled
strand becomes covalently bound to protein. The strand transfer
reaction was initiated by adding a 50-fold molar excess of an 18-mer
acceptor strand (either DNA or RNA) complementary to the 5
single-strand tail of the covalent donor complex (Fig. 3) while
simultaneously increasing the ionic strength to 0.3 M NaCl.
Addition of NaCl during the religation phase promotes dissociation of
the topoisomerase after strand closure and prevents recleavage of the
strand transfer product. Ligation of the covalently held 12-mer
CGTGTCGCCCTT to the 18-mer yields a 32P-labeled 30-mer
(Fig. 4, lane 1). The suicide intermediate
transferred 94% of the input CCCTT-containing strand to the 18-mer DNA
strand (Fig. 3). The extent of religation at the earliest time point (5 s) was 90% of the end point value. We calculated from this datum a
religation rate constant (krel) of >0.5
s
1. We had determined previously (from experimental
values for kcl and Keq at
37 °C) a krel value of ~1.3
s
1 (18).
Topoisomerase readily ligated the covalently held 12-mer DNA to an
18-mer RNA acceptor to form a 30-mer product (Fig. 4, lane 5). 89% of the input CCCTT-strand was transferred to RNA, with 40% of the end point value attained in 5 s. We used this datum to
estimate a rate constant of 0.1 s1 for single-turnover
strand transfer to RNA. Thus, religation to DNA was about 10 times
faster than religation to RNA. The slowed rate of RNA religation is
likely to account for the observed increase in the cleavage-religation
equilibrium constant (Keq = kcl/krel) on the
DNA-p-RNA 36-mer.
The
predicted product of strand transfer to RNA is a 30-mer tandem
DNA-RNA strand
(5-CGTGTCGCCCTTAUUCCGAUAGUGACUACA) uniquely 32P-labeled at the 5
end. The structure of this molecule
was confirmed by analysis of the susceptibility of this product to
treatment with NaOH. The labeled 30-mer RNA ligation product was
converted nearly quantitatively into a discrete species that migrated
more rapidly than the input 18-mer CCCTT-containing DNA strand (Fig. 4,
lane 6). The mobility of this product was consistent with a chain length of 13 nucleotides. The expected 32P-labeled
alkaline hydrolysis product of the RNA strand transfer product is a
13-mer (5
-CGTGTCGCCCTTAp). Control reactions showed that
neither the 32P-labeled 18-mer scissile strand of the
suicide substrate nor the 30-mer product of strand transfer to DNA was
susceptible to alkali (Fig. 4, lanes 4 and 2). We
conclude that topoisomerase can be used to ligate RNA to DNA
in vitro.
Practical applications of
topoisomerase-mediated strand transfer to RNA include the 5
tagging of RNA transcripts. Bacteriophage RNA polymerases have been
used widely to synthesize RNA in vitro from plasmid DNA
templates containing phage promoters. To test whether such transcripts
were substrates for topoisomerase-catalyzed ligation, we constructed a
CCCTT-containing suicide cleavage substrate that, when cleaved by
topoisomerase, would contain a 5
single-strand tail complementary to
the predicted 5
sequence of any RNA transcribed by T3 RNA polymerase
from a pBluescript vector (Fig. 5). A 36-nucleotide T3
transcript was synthesized in a transcription reaction containing [
32P]GTP. The RNA was treated with alkaline
phosphatase to dephosphorylate the 5
terminus. The topoisomerase-DNA
covalent intermediate was formed on an unlabeled suicide substrate.
Incubation of the radiolabeled T3 transcript with the suicide
intermediate resulted in the conversion of the 36-mer RNA into a novel
species that migrated more slowly during polyacrylamide gel
electrophoresis (not shown). The apparent size of this product (48 nucleotides) was indicative of ligation to the 12-mer CCCTT DNA strand.
The kinetics of DNA ligation to the T3 transcript are shown in Fig. 5.
The reaction was virtually complete within 1 min; at its end point,
29% of the input RNA had been joined to DNA. No DNA-RNA ligation
product was formed in reaction containing a T3 transcript that had not
been treated with alkaline phosphatase (not shown).
Formation of Insertions and Deletions: A Kinetic Analysis
The
acceptor polynucleotides used in the experiments described above were
capable of hybridizing perfectly with the 5 single-strand tail of the
topoisomerase-DNA donor complex. It had been shown previously that the
vaccinia virus topoisomerase is capable of joining the CCCTT-strand to
an acceptor oligonucleotide that hybridizes so as to leave a single
nucleotide gap between the covalently bound donor 3
end and the 5
terminus of the acceptor. Religation across this gap generated a 1-base
deletion in the product compared with the input scissile strand (5).
The enzyme also catalyzes strand transfer to an acceptor
oligonucleotide that, when hybridized, introduces an extra nucleotide
between the donor 3
end and the penultimate base paired nucleotide of
the acceptor. Religation in this case will produce a 1-base insertion
(5). Deletion and insertion formation in vitro have also
been documented for mammalian type I topoisomerase (19). However, there
has been no report of the effects of acceptor strand gaps and
insertions on the rate of strand joining by these
enzymes.
We assessed the kinetics of strand transfer by the vaccinia
topoisomerase covalent intermediate to acceptor oligonucleotides that
base pair with the donor complex to form either a fully base-paired 3
duplex segment or 3
duplexes with a 1- or 2-nucleotide gap. 84% of
the input DNA substrate was ligated to the fully paired acceptor in
10 s, the shortest time analyzed (Fig.
6A). The size of the strand transfer product
was 30 nucleotides, as expected (Fig. 7, lane
3). No 30-mer product was formed in the absence of the added
acceptor strand (Fig. 7, lane 2).
Religation across a 1-nucleotide gap was highly efficient, albeit slow.
85% of the input substrate was joined across a 1-nucleotide gap to
yield the expected 29-nucleotide product (Fig. 6A and Fig. 7, lane 4). The kinetic data in Fig. 6 fit well to a single
exponential with an apparent rate constant of 0.005 s1.
Thus, single-turnover strand closure by topoisomerase across a
1-nucleotide gap was 2 orders of magnitude slower than the rate of
joining across a fully paired nick. Vaccinia topoisomerase catalyzed
strand transfer across a 2-nucleotide gap to form the anticipated
28-nucleotide product (Fig. 7, lane 5), but this reaction was feeble (Fig. 6A). We observed linear accumulation of the
2-nucleotide gap product over a 2-h incubation, at the end of which
only 10% of the input DNA had been joined. We estimated, based on the
initial rate, that religation across the 2-nucleotide gap was 2 orders of magnitude slower than joining across a 1-nucleotide gap (and hence 4 orders of magnitude slower than the rate of joining across a nick).
Similar experiments were performed using DNA acceptors that contained
either 1 or 2 extra nucleotides at their 5 ends (Fig. 6B).
Religation to these acceptors yielded labeled strand transfer products
of 31 and 32 nucleotides, respectively (Fig. 7, lanes 6 and
7). 90% of the input DNA was religated to form the
1-nucleotide insertion product (Fig. 6B). We calculated a
rate constant of 0.04 s
1 for religation with 1-nucleotide
insertion. A similar end point was achieved in the formation of a
2-nucleotide insertion product, but the strand transfer rate was
considerably slower (Fig. 6B). The observed rate constant
for 2-nucleotide insertion was 0.001 s
1, i.e.
3 orders of magnitude lower than krel at a
nick.
We examined strand transfer by topoisomerase to a set of
18-mer acceptors that were capable of forming base pairs with the 5
tail of the donor complex from positions
2 to
18 (relative to the
scissile +1 T:A base pair of the CCCTT element) but that have a base
mismatch at the
1 position immediately 3
of the scissile bond. The
control acceptor, which has a normal
1 A:T base pair, reacted to
completion in 10 s; 89% of the end point was achieved in 5 s
(Fig. 8). DNAs containing T:T, C:T, or G:T mispairs at
the
1 position supported the same extent of strand transfer; 77% of
the end point was attained in 5 s in each case (Fig. 8). Thus,
within the limits of detection of this experiment, mismatch at the
1
position had little effect on the strand transfer reaction. There are
clear and instructive differences between the effects of base
mismatches versus a single nucleotide deletion on the
rate of the strand joining step.
Kinetics of Intramolecular Hairpin Formation
In the
absence of an exogenous acceptor oligonucleotide, the 5-OH terminus of
the nonscissile strand of the 12-mer/30-mer covalent complex can flip
back and act as the nucleophile in attacking the DNA-(3-phosphotyrosyl)
bond (5). The reaction product is a hairpin molecule containing a 12-bp
stem and an 18-nucleotide loop. The kinetics of this reaction were
examined under single turnover conditions. In the experiment shown in
Fig. 9A, 65% of the input CCCTT strand was
converted to hairpin product in 3 h. The observed rate constant
was 5.7 × 10
4 sec
1. In parallel, we
analyzed the rate of hairpin formation by the covalent complex formed
on an 18-bp cleavage substrate (Fig. 9A). In this case, an
attack by the 5
-OH of the nonscissile strand yielded a hairpin
molecule containing a 12-bp stem and a 6-nucleotide loop. 69% of the
input CCCTT strand was converted to hairpin product in 10 h. The
observed rate constant was 8.2 × 10
5
sec
1. Thus, the 18-nucleotide 5
tail was ~7 times more
effective than the 6-mer 5
tail as the attacking nucleophile for
strand transfer in cis. Note that hairpin formation by these
covalent complexes occurs without any potential for base pairing by the single-strand tails.
To examine the contribution of base pairing to the rate of religation,
we altered the 5 terminal and penultimate bases of bottom strand of
the 18-mer/30-mer substrate to 5
-AT (Fig. 9B). Now, the
5
-terminal three bases of the bottom strand (5
-ATT) are identical to
the 5
-terminal bases of the leaving strand (5
-ATTCCC); hence, the
single-strand tail is self-complementary and capable of forming three
base pairs adjacent to the scissile phosphate. Intramolecular hairpin
formation on this DNA was extremely fast; the reaction was complete in
10-20 s (Fig. 9B). The observed religation rate constant
was 0.2 s
1. By comparing this value to the religation
rate constant on the noncomplementary 18-mer/30-mer substrate (Fig.
9A), we surmise that three base pairs accelerated the
reaction ~350-fold.
The 42-nucleotide 5 32P-labeled hairpin
product was gel-purified and tested as a substrate for covalent adduct
formation by the vaccinia topoisomerase. 55% of the input
radioactivity was transferred to the topoisomerase polypeptide in
15 s at 37 °C; an end point of 90% transfer was attained in
60 s (data not shown). The apparent rate constant for cleavage of
the hairpin was 0.06 s
1. Thus, the topoisomerase rapidly
and efficiently cleaved a CCCTT-containing molecule in which there were
no standard paired bases downstream of the scissile phosphate. The
hairpin cleavage rate constant is about one-fifth of
kcl on the 18-mer/30-mer suicide substrate, which contains five paired bases of duplex DNA 3
of the CCCTT site.
Vaccinia topoisomerase catalyzes a diverse repertoire of strand transfer reactions. Religation of the covalently bound DNA to a perfectly base-paired acceptor DNA oligonucleotide provides a model for the strand closure step of the DNA relaxation reaction. Here, we have analyzed the kinetics of strand transfer to alternative nucleic acid acceptors. Our findings provide new insights into the parameters that affect transesterification rate, illuminate the potential for topoisomerase to generate mutations in vivo, and suggest practical applications of vaccinia topoisomerase as an RNA modifying enzyme.
Sugar Specificity for Covalent Adduct Formation Resides within the CCCTT ElementVaccinia topoisomerase is apparently incapable of
binding covalently to CCCUU-containing RNA strands. This is the case
whether the CCCUU strand is part of an RNA-RNA or an RNA-DNA duplex
(9). We have now shown that the sugar specificity of the enzyme is attributable to a stringent requirement for DNA on the 5 side of the
scissile phosphate, i.e. the CCCTT site must be DNA.
Moreover, the CCCTT element must be a DNA-DNA duplex, because earlier
experiments showed that a CCCTT strand is not cleaved when it is
annealed to a complementary RNA strand (9). The RNA-DNA hybrid results are informative, because they suggest that the CCCTT site must adopt a
B-form helical conformation to be cleaved. RNA and DNA polynucleotide
chains adopt different conformations within an RNA-DNA hybrid, with the
RNA strand retaining the A-form helical conformation (as found in
double-stranded RNA), whereas the DNA strand adopts a conformation that
is neither strictly A nor B, but is instead intermediate in character
between these two forms (20, 21). Vaccinia topoisomerase makes contacts
with the nucleotide bases of the CCCTT site in the major groove (9,
22). It also makes contacts with specific phosphates of the CCCTT site
(23). Adoption by the CCCTT site of a non-B conformation may weaken or
preclude these contacts.
Our finding that vaccinia topoisomerase is relatively insensitive to the nucleotide sugar composition downstream of the scissile phosphate implies that the conformation of the helix in this portion of the ligand is not important for site recognition or reaction chemistry. Topoisomerase cleaves DNA-p-RNA strands in which the leaving strand is RNA. Indeed, the extent of cleavage at equilibrium is significantly higher than that achieved on a DNA-p-DNA strand.
Strand Transfer to RNAThe increase in the
cleavage-religation equilibrium constant Keq (=
kcl/krel) on the
DNA-p-RNA substrate can be explained by our finding that the rate of
single-turnover RNA religation krel(RNA) is
about one-tenth that of krel(DNA). Nonetheless,
the extent of religation to RNA is quite high,
i.e. ~90% of the input CCCTT strand is religated to an
18-mer RNA acceptor strand in a 2-min reaction. We have shown that a
CCCTT-containing DNA strand can be rapidly joined by topoisomerase to a
transcript synthesized in vitro by bacteriophage RNA
polymerase; ~30% of the RNA was transferred to the DNA strand in a
2-5 min reaction. This property can be exploited to 5 tag any RNA for
which the 5
terminal RNA sequence is known, i.e. by
designing a suicide DNA cleavage substrate for vaccinia topoisomerase
in which the nonscissile strand is complementary to the 5
sequence of
the intended RNA acceptor. Some practical applications include: (i)
32P-labeling of the 5
end of RNA; and (ii) affinity
labeling the 5
end of RNA, e.g. by using a biotinylated
topoisomerase cleavage substrate. A potential advantage of
topoisomerase-mediated RNA strand joining (compared with the standard
T4 RNA ligase reaction) is that ligation by topoisomerase can be
targeted by the investigator to RNAs of interest within a complex
mixture of RNA molecules.
It was reported earlier
that vaccinia topoisomerase can religate to complementary DNA acceptors
containing recessed ends or extra nucleotides, thereby generating the
equivalent of frameshift mutations (5). Similar reactions have been
described by Henningfeld and Hecht (19) for the cellular type I
topoisomerase. A key question is whether these aberrant religation
reactions are robust enough to implicate topoisomerase as a potential
mutagen in vivo. Our kinetic analysis suggests that they are
and provides the first clue as to what spectrum of frameshift reactions
is most likely to occur (taking into account only the intrinsic
properties of the topoisomerase). For the vaccinia enzyme, the
hierarchy of frameshift generating religation reactions is as follows:
+1 insertion > 1 deletion > +2 insertion
2
deletion.
The slowest of these topoisomerase-catalyzed reactions is strand
closure across a 2-nucleotide gap (initial rate = 0.002% of input
DNA religated/sec). In this situation, the attacking nucleophile is
held in place at some distance from the DNA-protein phosphodiester by
base pairing with the nonscissile strand. Moving the 5-hydroxyl 1 base
pair closer to the phosphodiester enhances reaction rate by a factor of
100. Extra nonpaired nucleotides appear to pose much less of an
impediment to strand joining to form 1- or 2-nucleotide insertions. The
active site of the topoisomerase may be able to accommodate
extrahelical nucleotides; alternatively, these nucleotides may
intercalate into the DNA helix at the topoisomerase-induced nick.
There are two potential pathways for topoisomerase to form minus
frameshifts in vivo, which differ as to how the acceptor strand is generated: (i) the 5 end of the leaving strand can be
trimmed by a nuclease, after which ligation could occur across the
resulting gap; or (ii) a homologous DNA single strand could attack the
covalent intermediate. The second pathway presumably requires a
helicase to form the invading strand (and perhaps also to displace the
leaving strand). In the case of plus frameshifts, only the latter
pathway would be available to the topoisomerase, i.e.
because no mechanism exists to add nucleotides to the 5
terminus of
the original leaving strand. No matter which pathway is taken, it is
reasonable to assume that the most rapidly catalyzed mutagenic
strand-joining reactions are the ones most likely to make their mark
in vivo. If the religation reaction is slow, as for
2
frameshifting, then the cell has greater opportunity to repair the
mutagenic lesion, e.g. by removing the covalently bound topoisomerase. This could entail: (i) excision of a patch of the DNA
strand to which the topoisomerase is bound; or (ii) hydrolysis of the
topoisomerase-DNA adduct. An enzyme that catalyzes the latter reaction
was discovered recently by Yang et al. (24).
Introducing a base mismatch at the 1 position immediately flanking
the scissile phosphate has almost no effect on the rate of religation.
This result is in stark contrast to the 10
2 rate effect
of a 1-nucleotide gap. We infer that the
1 base mismatches do not
significantly alter the proximity of the 5
-hydroxyl nucleophile of the
terminal nucleotide to the scissile phosphate at enzyme's active site.
Our results indicate clearly that topoisomerase has the capacity to
generate missense mutations in vitro. The single-strand
invasion pathway invoked above for frameshift mutagenesis could, in
principle, provide the opportunity for topoisomerase to create missense
mutations in vivo. The kinetics of ligation in
vitro suggest that topoisomerase-generated missense mutations would predominate over frameshifts.
Kinetic analysis
of intramolecular hairpin formation by the vaccinia topoisomerase
provides the first quantitative assessment of the role of base
complementarity in strand closure. The rate constant for attack on the
DNA-(3-phosphotyrosyl) bond by a nonpairing 18-nucleotide single
strand linked in cis to the covalent complex was 5.7 × 10
4 sec
1. Altering only the terminal bases
of the single-strand tail to allow the formation of base pairs at the
1,
2, and
3 positions increased the rate constant for hairpin
formation by 350-fold. The rate of religation in cis with 3 potential base pairs was nearly the same as the rate of religation to a
non-covalently linked acceptor strand that forms 18 base pairs 3
of
the scissile bond. The ability of the covalently bound enzyme to take
up and rapidly rejoin DNA strands with only three complementary
nucleotides lends credence to the suggestion that vaccinia
topoisomerase catalyzes the formation of recombination intermediates
in vivo (25), either via strand invasion or by reciprocal
strand transfer between two topoisomerase-DNA complexes. Efforts to
model these reactions in vitro are under way.