In the Laboratory
DNA Topology Analysis in the Undergraduate Biochemistry Laboratory
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Michael V. Keck Division of Physical Sciences, Emporia State University, Emporia, KS 66801;
[email protected] A regular point of difficulty for many students in an introductory biochemistry class is the concept of DNA topology and the relationships among DNA topoisomers. It is an important concept, however, because proper DNA winding and topology is critical for many intracellular processes, such as replication, transcription, recombination, and repair. And yet, even the relatively simple topology of supercoiled circular DNA is often found to be confusing. This Journal has published articles on DNA supercoiling (1) and its biological significance (2), which provide excellent background on the subject. I find that the laboratory experiments described here, which I use in my junior–senior-level introductory biochemistry class, greatly enhance the students’ comprehension. A graduate student studying DNA structure would also benefit. Background It is well known that the degree of helical winding and overall DNA topology are intimately and inextricably related (1–3). DNA made in cells is typically underwound. The reasons for this are several, but include packaging effects as well as energetic advantages during DNA replication and transcription. The unwinding is manifest largely through supercoiling, wherein the DNA winds upon itself (or around proteins such as histones). Such superhelical winding allows the DNA to maintain a normal helical twist of approximately 10 base pairs per turn. Small circular DNAs of extrachromosomal origin, such as plasmids, are also underwound (2), and provide a relatively simple system for study. When describing plasmid DNA topology, the key concept is that the sum of the number of duplex turns (β) plus the number of superhelical turns (τ) (the number of times the duplex axis wraps around the superhelical axis) must remain constant as long as both DNA strands remain intact. This condition is often expressed through eq 1, where the constant parameter α is termed the linking number. α=β+τ
(1)
For DNA that is underwound, τ will be negative. The underwound, negatively supercoiled DNA circle is in a state of torsional stress, and any process that relieves this stress is thermodynamically favorable. There are two obvious ways to remove these supercoils and relieve the torsional stress. The first is to cleave one or both of the DNA strands. If only one strand is cleaved, the DNA is said to be nicked. Nicked DNA is still circular, because one strand remains intact. If both strands are cleaved directly opposite each other, the DNA is said to be cut, and a linear DNA is generated. When DNA is nicked or cut, the topological constraint in eq 1 is relieved. Many chemical agents are capable of promoting such chemistry. Examples include Fe(II)–EDTA (4) and Cu(I)–o-phenanthroline (5), which are used in DNA mapping studies, chemotherapeutics
such as bleomycin (6 ), and the ene-diyne antibiotics as exemplified by calicheamicin (7 ). Additionally, a group of enzymes known as topoisomerases can relieve the torsional stress by removing supercoils one or two at a time, thereby generating covalently closed relaxed circular DNA. These enzymes function by introducing a transient nick or cut into the DNA, passing one or two strands through the nick/cut, then resealing the DNA (8). The second way to remove supercoils from circular DNA is to allow it to bind to an unwinding agent. The prototypical DNA unwinding agent is the ethidium cation, a heteroaromatic phenanthridine derivative that binds by intercalation. The chemotherapeutic agent cis-diamminedichloroplatinum (cisplatin), which binds covalently to DNA primarily through the N-7 positions of adjacent guanines, is also known to unwind DNA (9). Since the DNA is not nicked or cut during this process, the topological restraints remain, and the helical unwinding is compensated by a removal of negative supercoils. That DNA unwinding will remove negative supercoils can be readily understood from eq 1. Unwinding the double helix will result in a decrease in the helical twist (β). Because α must remain constant, τ must increase; and because τ is a negative number, an increase causes its value to tend toward zero, indicating the removal of the (negative) supercoils. Agarose gel electrophoresis is a simple method for evaluating the topological state of circular DNA. The more highly supercoiled the circular DNA molecule is, the more compact its structure and the faster it migrates in the gel, thus making the various topological forms readily resolvable. Electrophoresis therefore provides an ideal tool to study the topological effects of DNA unwinding and cleavage. To help students better understand the topology of circular DNA, I have developed a set of experiments that utilize agarose gel electrophoresis to study the phenomena discussed above. The first experiment uses a copper–o-phenanthroline complex to nick the DNA and induce transitions between the three extreme topological forms. The second experiment uses cisplatin to unwind the DNA and cause a gradual transition from a supercoiled molecule to one without supercoils. The third experiment utilizes the enzyme topoisomerase I to generate a set of topoisomers from a supercoiled DNA substrate. The three are presented here as a set because I have found that, when considered together, they are more instructive for students than when considered individually. Each can, however, serve as a stand-alone experiment to illustrate a specific point. Summary of Materials and Methods
Materials Any supercoiled circular DNA is suitable for this experiment. We typically use φ X174 RF DNA, as it can be purchased relatively cheaply. Topoisomerase I is the most expensive reagent, at a cost of around $100 for 200 units, a quantity that is
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In the Laboratory
enough for 20 or more experiments. cis-Diamminedichloroplatinum (cis-DDP or cisplatin) can be purchased at a cost of around $60 for 250 mg, a quantity that is enough for several years. All other reagents and buffers are commonly found in chemistry or biology departments. Additional details and recipes are provided online.W
Methods W DNA cleavage using Cu–(OP)2. Cu–(OP)2 at concentrations ranging from 0 to 10 µM is allowed to react with 0.2-µg quantities of φ X174 RF DNA. Reactions (10 µL total volume) are carried out in 5 mM phosphate buffer, pH 7.4, in the presence of 1.0 mM hydrogen peroxide and 1.0 mM DTT. Reactions are allowed to proceed for 15–30 min at various temperatures, then analyzed by agarose gel electrophoresis. Reaction of DNA with cisplatin. A 0.3-µg quantity of φ X174 RF DNA is allowed to react with cis-diamminedinitratoplatinum (prepared in advance by the instructor from cis-DDP and silver nitrate) at Pt–nucleotide ratios (often termed rf ) that range between 0.02 and 0.80. The reactions are carried out in 15 µL of TE buffer for 2–3 h at 37 °C, then quenched by addition of NaCl to a concentration of 0.1 M. Results are analyzed by 1% agarose gel electrophoresis (without ethidium). Reaction of DNA with topoisomerase I. The procedure is essentially that described by Keller (10). A 1.0-µg portion of DNA is incubated with 10 U of topoisomerase I at 0 °C for 1–5 min in TNE buffer, pH 7.9. The reaction is quenched by the addition of SDS to 1%, then analyzed by 1.4% agarose gel electrophoresis at 30–40 V overnight. This experiment can be carried out concurrently with the cisplatin experiment and analyzed on the same gel. Hazards Two reagents used in these experiments should be particularly noted. Ethidium bromide is a potent mutagen, typically sold as a fine powder. It is therefore suggested that the instructor or teaching assistant prepare the 10-mg/mL solution for the students in advance, while wearing gloves and mask. Students should wear gloves while handling the solution, and special care should be taken to clean all spills immediately and thoroughly. Used ethidium should be reacted with bleach before disposal. Cisplatin is also a mutagen and carcinogen, and gloves should be worn when using it. Results and Discussion Typical results for these experiments are shown in Figures 1 through 3. In the OP–Cu-mediated DNA cleavage experiment (Fig. 1), the conversion of supercoiled (form I) to nicked relaxed (form II) DNA is indicated by reciprocal change in the relative intensities of these two bands as a function of Cu–(OP)2 concentration. The appearance of linear DNA (form III; migrates between the supercoiled and nicked forms) can often be observed at higher temperatures and concentrations. Linear DNA is formed because, as the number of nicks increases, so does the probability that two independently formed nicks will occur directly opposite each other. The formation of two such nicks is the functional equivalent of a double-stranded cut. Form III DNA begins to appear in lanes 1472
4 and 9. Lane 5 indicates almost complete loss of the nicked DNA, as well as some “smearing” below the form III band, which is the result of further nonspecific DNA degradation that generates smaller fragments. In lane 10 (highest copper concentration and temperature), the DNA has been degraded to such an extent that it is barely visible on the gel. The binding of cisplatin to DNA (Fig. 2) alters the migration rate of the supercoiled plasmid as a result of DNA unwinding, which removes supercoils (see above). As the amount of bound platinum increases, migration rates gradually decrease, to the point at which all the negative supercoils have been removed. This is termed the coalescence point, as it occurs when the supercoiled band comigrates with the nicked band that is invariably present in plasmid DNA samples. Beyond this point, continued unwinding generates positive supercoils, and the mobility of the supercoiled DNA increases again. This is starting to occur in lane 14 of Figure 2. It is important to emphasize that in this experiment, the linking number remains unchanged; it is the relative amounts of helical and superhelical winding that are being altered by the drug. If further investigation is desired, the bound platinum– nucleotide ratio (r b) at the coalescence point can be determined, and this data can be used, along with the known unwinding
Figure 1. Reaction of covalently closed circular DNA with copper– o-phenanthroline. All reaction mixtures contained 0.2 µg of DNA and 1 mM hydrogen peroxide and dithiothreitol. Reactions in lanes 1–5 were carried out at room temperature; those in lanes 6–10 were carried out at 37 °C. Lanes 1 and 6, DNA control; lanes 2 and 7, 0.25 µM Cu(OP)2; lanes 3 and 8, 1.0 µM Cu(OP)2; lanes 4 and 9, 2.5 µM Cu(OP)2; lanes 5 and 10, 10.0 µM Cu(OP)2.
Figure 2. Reaction of covalently closed circular DNA with increasing concentrations of cisplatin. Formal Pt–nucleotide ratios are lane 1, 0; lane 2, 0.02; lane 3, 0.05; lane 4, 0.08; lane 5, 0.10; lane 6, 0.13; lane 7, 0.17; lane 8, 0.20; lane 9, 0.25; lane 10, 0.30; lane 11, 0.40; lane 12, 0.50; lane 13, 0.60; lane 14, 0.80.
Figure 3. Reaction of supercoiled circular DNA with topoisomerase I. All reactions were carried out at 0 °C using 1.0 µg of DNA. Lane 1, DNA control; lane 2, DNA + 10 U topoisomerase I, 1 min; lane 3, DNA + 10 U topoisomerase I, 5 min.
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In the Laboratory
angle for cisplatin of 13° (9), to estimate the superhelical density and the linking number of the plasmid. Details are provided with the supplemental information found online.W The ladder of topoisomers generated by topoisomerase I is very illustrative (Fig. 3). DNA topoisomerase I introduces a transient nick in the DNA by forming a covalent DNA– enzyme complex. The intact strand is then allowed to pass through this nick and the nick is resealed. The enzyme will repeat this process successively until the DNA is completely relaxed. Reaction conditions involving short reaction times and low temperatures result in a mixture of topoisomers that are readily resolved. In a carefully executed experiment, the number of supercoils can be estimated by counting bands. The critical concept in this experiment is that for each topoisomerase-mediated nicking and sealing cycle, the linking number changes by one. This is fundamentally different from the cisplatin experiment, where supercoils are removed by helix unwinding while keeping the linking number constant.
to a significantly lesser degree (11). Additionally, because of their integral roles in DNA replication and transcription, topoisomerases are actively pursued targets for rational drug design (12). Indeed, several currently used chemotherapeutic agents, such as adriamycin and topotecan, target topoisomerase or its DNA complex. The topic of DNA damage can be conveniently introduced through these experiments, which naturally leads to related topics such as mutagenesis, chemical carcinogenesis, and DNA repair. The subject of DNA repair is of intense current interest (13, 14), and in fact much work has been done concerning the repair of cisplatin–DNA adducts (15, 16 ). It is always a subject of great interest to my students. Finally, students are taught the common biochemical technique of DNA analysis by agarose gel electrophoresis through an interesting set of applications.
Advantages and Benefits of the Experiments
Detailed experimental procedures, student handouts, and instructor notes are available in this issue of JCE Online.
I find that this set of experiments, when considered as a whole, is useful to the students in several respects. Students are better able to understand the concept of DNA topology. The copper-mediated DNA cleavage experiment demonstrates the extreme topological forms of circular DNA. It is clear from the gels that the transformation from one form to the other occurs in a single step upon DNA strand cleavage, and the consequences of relieving the torsional stress are readily apparent. This exercise is followed by the platinum and topoisomerase experiments, which allow students to see the effect of removing supercoils gradually within a set of topological constraints. Supercoil removal by each of these three methods is fundamentally different. As a result of seeing these effects experimentally and subsequently being asked to explain them in the lab report, the student finishes this lab with a reasonably good understanding of topological phenomena and interrelationships, as well as some of the factors that can affect them. All three experiments are readily related to medicinal chemistry applications. The mechanism of DNA cleavage by Cu–(OP)2, which is initiated by hydrogen atom abstraction from the ribose ring, mimics the chemistry of several antitumor drugs, such as bleomycin or the ene-diyne antibiotics. (In fact, the Cu–(OP)2 experiment described here may be carried out with bleomycin, albeit at greater cost.) Cisplatin is currently used in many chemotherapeutic regimens. An interesting elaboration of this experiment, which I have done in the past, is to compare the DNA unwinding caused by cisplatin with that of the inactive trans isomer, which unwinds
WSupplemental
Material
Literature Cited 1. Scovell, W. M. J. Chem. Educ. 1986, 63, 562–565. 2. Sinden, R. R. J. Chem. Educ. 1987, 64, 294–301. 3. See, for example Garrett, R. H.; Grisham, C. M. Biochemistry, 2nd ed.; Saunders: Philadelphia, 1999; pp 375–378. 4. Hertzberg, R. P.; Dervan, P. B. Biochemistry 1984, 23, 3934–3945. 5. Thederahn, T. B.; Kuwabara, M. D.; Larsen, T. A.; Sigman, D. S. J. Am. Chem. Soc. 1989, 111, 4941–4946. 6. Hecht, S. M. In Cancer Chemotherapeutic Agents; Foye, O. W., Ed.; ACS Professional Reference Book Series; American Chemical Society: Washington, DC, 1995; Chapter 10. 7. Lee, M. D.; Ellestad, G. A.; Borders, D. B. Acc. Chem. Res. 1991, 24, 235–243. 8. Wang J. C. Annu. Rev. Biochem. 1996, 65, 635–692. 9. Bellon, S. F.; Coleman, J. H.; Lippard, S. J. Biochemistry 1991, 30, 8026–8035. 10. Keller, W. Proc. Natl. Acad. Sci. USA 1975, 72, 2550–2554. 11. Keck, M. V.; Lippard, S. J. J. Am. Chem. Soc. 1992, 114, 3386–3390. 12. Chen A. Y.; Liu L. F. Annu. Rev. Pharmacol. Toxicol. 1994, 34, 191–218. 13. Sancar, A. Annu. Rev. Genet. 1995, 29, 69–105. 14. Wood, R. D. Annu. Rev. Biochem. 1996, 65, 135–167. 15. Zamble, D. B.; Mu, D.; Reardon, J. T.; Sancar, A.; Lippard, S. J. Biochemistry 1996, 35, 10004–10013. 16. Huang, J.-C.; Zamble, D. B.; Reardon, J. T.; Lippard, S. J.; Sancar, A. Proc. Natl. Acad. Sci. USA 1991, 91, 10394–10398.
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