From the
The widespread application of polymerase chain reaction and
related techniques in biology and medicine has led to a heightened
interest in thermophilic enzymes of DNA metabolism. Some of these
enzymes are stable for hours at 100 °C, but no enzymatic activity
on duplex DNA at temperatures above 100 °C has so far been
demonstrated. Recently, we isolated topoisomerase V from the
hyperthermophile Methanopyrus kandleri, which grows up to 110
°C. This novel enzyme is similar to eukaryotic topoisomerase I and
acts on duplex DNA regions. We now show that topoisomerase V catalyzes
the unlinking of double-stranded circular DNA at temperatures up to 122
°C. In this in vitro system, maximal DNA unlinking occurs
at 108 °C and corresponds to complementary strands being linked at
most once. These results further imply that in the presence of
sufficient positive supercoiling DNA can exist as a double helix even
at 122 °C.
Hyperthermophiles are globally distributed in such diverse
environments as deep ocean vents, geothermal springs, and hot oil
reservoirs
(1, 2, 3, 4) . They are not
just scientific curiosities, but are also potential sources of
thermostable enzymes for scientific and industrial
purposes
(1, 2, 3, 4, 5, 6, 7) .
Evolutionary studies indicate that some hyperthermophiles may be the
closest relatives of eukaryotic organisms (8-10). An increasing
number of facts support the idea that life on this planet originated at
much higher temperature than was thought before, and that
hyperthermophiles are contemporary relics of the most ancient
cells
(11, 12, 13, 14, 15, 16) .
The highest temperature at which cells grow under laboratory
conditions is 110 °C
(17, 18) . The activity of
proteases and some other enzymes has been detected at 120-140
°C
(19, 20, 21) . However, the disruption of
DNA secondary structure and destabilization of its chemical bonds when
the temperature exceeds 100 °C complicate the in vitro studies of hyperthermophilic proteins acting on DNA. For example,
hyperthermophilic DNA polymerases are remarkably stable but have no
activity in vitro at and above 100
°C
(22, 23, 24) . Similarly, reverse gyrase
and topoisomerase III (both requiring single-stranded regions of DNA as
a substrate) are apparently inhibited by the accumulation of denatured
ssDNA
Topoisomerase V, a recently discovered novel hyperthermophilic
enzyme (15, 16), relaxes both positively and negatively supercoiled DNA
at temperatures below 90 °C by a mechanism similar to that of
eukaryotic topoisomerase I
(29, 30) . At 90-100
°C, above the melting temperature of DNA, it unlinks circular DNA.
This DNA unlinking reaction mimics negative supercoiling by gyrase but
is ATP-independent. Because topological constraint prevents covalently
closed DNA from complete denaturation even above 100
°C
(31, 32) , we expected that topoisomerase V might
be active under these conditions, with the remaining duplex regions in
the circular DNA available as its substrate.
At temperatures near 100 °C, DNA melting, degradation,
and the accumulation of denatured ssDNA
(15, 26, 27) inhibit DNA processing enzymes. Topoisomerase V, like other
enzymes, is inhibited by ssDNA at very high temperature. This is
illustrated in Fig. 1by varying the ratio of M13 ssDNA to pBR322
dsDNA (lanes 3-5). In the absence of M13 ssDNA at 95
°C, topoisomerase V unlinks relaxed pBR322 and, like gyrase,
produces moderately supercoiled topoisomers (lane 3). The
inhibition is proportional to the ssDNA:dsDNA weight ratio
(approximately 50% at the 1:1 ratio), and is the same whether the
enzyme is preincubated with ssDNA or dsDNA (not shown). Hence the
enzyme reversibly binds to ssDNA and dsDNA without preference for
either one. This result indicates that during prolonged incubation at
very high temperature covalently closed plasmid molecules would break,
denature, and become inhibitory for topoisomerase V.
Fig. 1
and our previous data
(15, 16) demonstrate that betaine and glutamate minimize inhibitory
interaction of topoisomerase V with ssDNA and that thermal degradation
of DNA is decreased at high salt concentration. We further determined
that the optimal concentrations of betaine and potassium glutamate are
1.1 and 1.5 M, respectively (data not shown). Under these
conditions at temperatures in the range 100-122 °C,
topoisomerase V unlinks relaxed DNA in 90 s (Fig. 2).
Electrophoretic mobility of the products obtained at 100 °C and
116-122 °C coincides with that of native negatively
supercoiled pBR322. The mobility of products obtained at temperatures
between 100 °C and 116 °C is higher and reaches maximum at 108
°C. Degradation of the substrate dsDNA (lane 2) is minimal
even at 110 °C due to the high salt concentration. However,
approximately 50% of the unwound product (lane 9) degraded
during incubation and migrated as a highly diffuse band ahead of the
intact DNA circles (not shown). At 122 °C the band corresponding to
intact unwound circles is still visible, although the degradation of
DNA is substantial (lane 14). Although DNA degradation was
minimal at 110 °C for these experiments, it was more extensive for
longer incubation periods (data not shown).
Construction of an in vitro system for unlinking of circular DNA at temperatures above 100
°C makes it possible to start to identify those factors responsible
for DNA stabilization inside hyperthermophiles. The ability of
topoisomerase V to unlink DNA at high temperature may make it extremely
useful for thermal cycling processes in which topological constraint
inhibits normal enzymatic processes.
We thank Regis Krah and Marty Gellert for
encouragement, reviewing the manuscript, and many valuable suggestions;
Sergey Ryazantsev and Michael Hiykinson for help with platinum
shadowing; and Margaret Kowalczyk for the artwork.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
at high
temperature
(25, 26, 27, 28) .
Materials
Methanopyrus kandleri topoisomerase V was prepared as in Ref. 16. Escherichia coli RecA protein was from Boehringer Mannheim.
Inhibition of Topoisomerase V by ssDNA
2 units of
topoisomerase V was mixed with 0.1 µg of relaxed circular pBR322
dsDNA and M13 ssDNA in 30 mM Tris-HCl (pH 8 at 25 °C), 0.3
M NaCl with or without betaine and incubated at 95 °C for
15 min. The results were analyzed by 1.5% agarose gel electrophoresis.
DNA Unlinking above 100 °C
0.1 µg of
relaxed pBR322 DNA was incubated in 30 mM Tris-HCl (pH 8), 1.5
M potassium glutamate, 10 mM magnesium acetate, 1.1
M betaine with 10 units of topoisomerase V at the appropriate
temperature for 90 s. To prevent boiling, the reaction mixture of 4
µl was placed inside a 25-µl fast protein liquid chromatography
sample loop and closed by domed nuts.Electron Microscopy-pBR322 DNA was incubated with topoisomerase V
in 30 mM Tris-HCl (pH 8), 1.5 M potassium glutamate,
1.1 M betaine at 106 °C for 5 min. The recovered DNA was
denatured by glyoxal treatment at 62 °C for 30
min
(33, 34) , desalted, and treated with exonuclease
VII. The products were mixed with RecA protein (1:40 mass ratio)
in 50 µl containing 25 mM Tris-HCl (pH 7.6), 2.5
mM MgCl
, and 0.5 mM ATP
S. After a
30-min incubation at 37 °C, the samples were prepared for electron
microscopy using the single carbon method with some
modifications
(35, 36, 37) . Grids then were
stained with 0.2-0.5% uranyl acetate and both rotatory and
unidirectionally (at a 10° angle and a source-to-sample distance of
10 cm) shadowed with platinum.
Figure 1:
Inhibition of topoisomerase V activity
by ssDNA and its prevention by betaine. Lane 1, control M13
ssDNA (C, circular, L, linear); lane 2,
control relaxed topoisomers of pBR322. Lanes 3-5,
topoisomerase V was mixed with relaxed circular pBR322 dsDNA and M13
ssDNA at the indicated weight:weight ratio and incubated at 95 °C.
The yield of unwound pBR322 products decreases upon the addition of M13
ssDNA. Lanes 6-8, the same as lanes 3-5,
but 2.2 M betaine was added to the reaction. The yield of
unwound pBR322 products is the same in lanes
6-8.
To prevent
ssDNA from inhibiting topoisomerase V, we searched for agents that
would allow topoisomerase V activity but block its binding to ssDNA.
Betaine
(38, 39, 40) proved to satisfy these
conditions. 2.2 M betaine completely prevents the inhibition
of topoisomerase V by ssDNA (Fig. 1, lanes 6-8).
Our data indicate that one of the unaccounted physiological roles of
betaine may be prevention of nonspecific protein-nucleic acid
interactions.
Figure 2:
DNA unlinking by topoisomerase V at
temperatures above 100 °C. Control pBR322 before (lane 1)
and after (lane 2) incubation without enzyme at 110 °C.
Lanes 3-14, relaxed pBR322 DNA was incubated with
topoisomerase V at the indicated temperatures. The topoisomers obtained
at 92-96 °C (lanes 3-5) are relaxed at those
temperatures and slightly negatively supercoiled at room temperature.
The products of topoisomerase V activity at 100-122 °C
(lanes 6-14) are the highly unwound topoisomers that
migrate as one band (U).
The composition of the
unlinked complexes, assayed by electron microscopy, is shown in
Fig. 3
. The principal product in the sample with the highest
electrophoretic mobility consists of pairs of concatenated ssDNA
circles linked once (isolated ssDNA circles and degraded molecules are
also present). From these data we estimated that the rate of DNA
unlinking at 108 °C is about 4 cycles/s/enzyme monomer, or about 16
times faster than the rate of DNA relaxation at 90 °C
(15) .
Electron microscopy of samples with lower electrophoretic mobility
showed that they lacked singly linked circles; instead, the DNA formed
small clumps (not shown), indicating incomplete unlinking
(41) .
We conclude that topoisomerase V can reduce the linking between
complementary strands down to a single link. Whether topoisomerase V
can completely unlink complementary strands is not known since isolated
ssDNA circles that can be observed may result from nicking the second
strand.
Figure 3:
Electron micrographs of DNA sample
maximally unlinked by topoisomerase V. Molecules shown here are
magnified approximately 120,000. Beneath each photograph is a tracing
of crossing complementary single-stranded rings showing the over- and
under-passing strand at each crossing. All shown ssDNA circles are
linked once.
We used formamide to study the relationship between DNA
denaturation and the ability of topoisomerase V to maximally unlink
DNA. Fig. 4shows that lowering the melting temperature of DNA by
formamide decreases the temperature T at which the
enzyme maximally unlinks DNA.
Figure 4:
The effect of formamide on the unlinking
reaction. The temperature for maximal unlinking of DNA by topoisomerase
V (T) decreases upon the addition of formamide.
T
and T
are, correspondingly,
the left and right positions of the arc of increased mobility of
unwound DNA (see Fig. 2 for details). The incubation was done
essentially as in Fig. 2, but 10 mM magnesium acetate was
replaced by 1 mM EDTA.
Our interpretation of the results is
shown in the model in Fig. 5. At temperatures below the DNA
melting temperature, topoisomerase V relaxes dsDNA. At higher
temperatures, when closed circular DNA melts, it creates regions of
positive supercoiling in which dsDNA is stabilized against further
melting. Topoisomerase V can relax this positive superhelicity,
i.e. decrease the linking number, and allow the DNA to melt
further. At temperatures near T (108 °C under
the conditions of Fig. 2), DNA can melt completely in the absence
of positive supercoiling and the enzyme allows the singly linked
molecule to be obtained. At still higher temperatures, a minimum amount
of positive supercoiling is required for any duplex formation. Since
dsDNA is the substrate for topoisomerase V, it cannot reduce the
linking number below this minimum and such DNA migrates in the gel more
slowly than the singly linked one.
Figure 5:
A schematic drawing illustrating the
products of topoisomerase V activity obtained at different
temperatures. ds and ssDNA regions are shown by thick and
thinlines, respectively.
Our analysis of topoisomerase V
activity suggests that positive superhelicity can stabilize the duplex
form of DNA even at temperatures up to 122 °C. Whether reverse
gyrase can create sufficient superhelicity to stabilize genomic DNA
in vivo at such high temperature remains unknown
(28) ,
but this represents a potential mechanism for maintaining the naked DNA
duplex at extremely hot conditions.
S, adenosine
5`-O-(thiotriphosphate).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.