©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
DNA Enzymology above 100 C
TOPOISOMERASE V UNLINKS CIRCULAR DNA AT 80-122 °C (*)

Sergei A. Kozyavkin (1)(§), Alexander V. Pushkin (3), Frederick A. Eiserling (3), Karl O. Stetter (2), James A. Lake (3), Alexei I. Slesarev (3)

From the (1) Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-0540, the (2) University of Regensburg, 8400 Regensburg, Federal Republic of Germany, and the (3) Molecular Biology Institute and Departments of Microbiology and Molecular Genetics and Biology, University of California, Los Angeles, California 90024

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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() at high temperature (25, 26, 27, 28) .

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.


EXPERIMENTAL PROCEDURES

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 ATPS. 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.


RESULTS AND DISCUSSION

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.


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.

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).


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.

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.


FOOTNOTES

*
This work was supported in part by grants from the National Science Foundation (to J. A. L.) and the Alfred P. Sloan Fellowship (to A. I. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Third Wave Technologies, 2800 S. Fish Hatchery Rd., Madison, WI 53711-5399. Tel.: 608-273-8933; Fax: 608-273-6989.

The abbreviations used are: ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; ATPS, adenosine 5`-O-(thiotriphosphate).


ACKNOWLEDGEMENTS

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.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.