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© 2005 by The International Union of Biochemistry and Molecular Biology Printed in U.S.A.

Laboratory Exercises

B IOCHEMISTRY AND M OLECULAR B IOLOGY E DUCATION

Vol. 33, No. 1, pp. 28 –33, 2005

Quantification of DNA by Agarose Gel Electrophoresis and Analysis of the Topoisomers of Plasmid and M13 DNA Following Treatment with a Restriction Endonuclease or DNA Topoisomerase I

Received for publication, June 8, 2004, and in revised form, July 20, 2004

John W. Tweedie‡ and Kathryn M. Stowell From the Institute of Molecular Biosciences, Massey University, Palmerston North, New Zealand

A two-session laboratory exercise for advanced undergraduate students in biochemistry and molecular

biology is described. The first session introduces students to DNA quantification by ultraviolet absorbance

and agarose gel electrophoresis followed by ethidium bromide staining. The second session involves treatment of various topological forms of DNA with a restriction endonuclease and with a DNA topoisomer- ase. This session introduces students to the concept of DNA topoisomers, to the properties of different forms of DNA, and to the activity of restriction endonucleases and topoisomerases toward these forms. The

exercise also involves measuring the size of linear duplex fragments of DNA by comparison of mobility with

a ladder of double stranded DNA of known sizes.

Keywords: DNA topoisomers, restriction endonuclease, DNA gel electrophoresis, DNA topoisomerases, DNA quantification.

When dealing with DNA there are two fundamental con- cepts that students in molecular biology must have a feeling for: the small quantities of DNA involved in most manipulations and the topological properties of these mol- ecules. These concepts are particularly relevant when ex- amining circular and linear DNA molecules of plasmids and bacteriophage by gel electrophoresis. Students must have an appreciation of the relation between the amount of DNA and its appearance after agarose gel electrophoresis and staining with ethidium bromide. They also need to be aware of the different mobilities exhibited by DNA mole- cules differing only in their topological form. In addition to these practical aspects, the role of DNA topology and its manipulation in DNA replication, transcription, and other metabolic transactions of DNA is fundamental to an un- derstanding of these topics. We have designed a two-part student laboratory exer- cise to illustrate and reinforce these concepts and to pro- vide an experimental background to lectures on DNA to- pology and topoisomerases in a third-year undergraduate course in the Institute of Molecular BioSciences. The the- oretical material is based on current biochemistry and molecular biology texts [1, 2]. About 500 students have used the exercise over the past 10 years. The first part of the experiment involves the quantifica- tion of plasmid DNA by ultraviolet (UV) 1 absorbance fol- lowed by agarose gel electrophoresis of serial dilutions of

‡ To whom correspondence should be addressed: Institute of Molecular Biosciences, Massey University, Palmerston North, New Zealand. E-mail: j.tweedie@massey.ac.nz. 1 The abbreviations used are: UV, ultraviolet; TAE, Tris acetate- EDTA; TE, Tris-EDTA; DTT, dithiothreitol.

the same DNA. This gives the students a feeling for the amount of DNA that can be visualized by this procedure. The experiment also introduces the concept of topoiso- mers because, in addition to covalently closed supercoiled circular DNA, most plasmid preparations also contain sub- stantial amounts of relaxed circles (generated by nicking of one strand) and smaller amounts of linear DNA generated by double-strand breaks. Students are faced with the sit- uation where a preparation, supposedly containing a sin- gle DNA species, shows two or three distinct bands on agarose gel electrophoresis. In the second part of the practical, plasmid and M13 DNA are treated with the restriction endonuclease EcoRI and eukaryotic DNA Topoisomerase I [3], and the reaction products are examined by agarose gel electrophoresis. Both covalently closed circular (RFI) and single-strand cir- cular (SS ) M13 DNA samples are treated. The results from this part of the practical allow the students to inter- pret and identify the multiple bands seen in gel electro- phoresis of plasmid DNA in the first part of the experiment. They can also interpret their results in terms of the ef- fects of Topoisomerase I and EcoRI on double-stranded and single-stranded DNA. The reading list for the prac- tical includes material on the functional aspects of DNA topology and its manipulation. This reinforces material on DNA topology contained in the corresponding lecture course. The experimental component of the exercise can be completed within two 3-h laboratory periods and is carried out by groups of two or three students. The total number of students accommodated is only limited by the availabil- ity of equipment for gel electrophoresis.

28 This paper is available on line at http://www.bambed.org

OUTLINE OF THE PRACTICAL

First Practical Session

Each group of students is provided with a sample of a plasmid with a single EcoRI site and which is about 3 kbp. We have used pUC118 for the data shown here. They are told the approximate concentration of the plasmid and the A 260 for double-stranded DNA. After calculating an appro- priate dilution they prepare a 1-ml aliquot, which is used to obtain A 260 and A 280 readings. They now calculate the actual DNA concentration of their sample and make a series of dilutions that will give known amounts of DNA per 10 l of TE buffer from 1 g down to 1 ng. Ten-microliter aliquots of the dilutions are mixed with 2 l of loading dye, and the mixed samples were loaded into the wells of an agarose gel. Following electrophoresis, the gels are stained with ethidium bromide, visualized under UV light, and the image recorded for return to the students for their interpretation.

Second Practical Session

In this session, the students are provided with samples of the plasmid used in the first session and samples of double-stranded circular (RFI) and single-stranded circular (SS ) DNA of the bacteriophage M13. Each group of students treats the covalently closed circular (ccc, RFI) and the single-stranded circular (SS ) DNA of bacteriophage M13 with DNA Topoisomerase I and with the restriction endonuclease EcoRI. Plasmid DNA (pUC118) is also treated with EcoRI and with DNA Topoi- somerase I. Following digestion, the various forms of M13 and pUC118 DNA are separated by agarose gel electro- phoresis in the absence of ethidium bromide. The gels are stained following electrophoresis and visualized and re- corded as for the first session.

MATERIALS AND METHODS

Buffers & Solutions—Tris acetate-EDTA (TAE) and Tris-EDTA (TE) buffers (10 ) are prepared as described by Sambrook and Russell [4]. Topoisomerase 1 buffer (10 ) contains Tris-HCl (0.5

M ), pH 7.5; KCl (0.5 M ); MgCl 2 (0.1 M ); EDTA (1.0 mM ); dithiothreitol (DTT) (5.0 mM ), and bovine serum albumin (300 g/ml). Topoi- somerase storage buffer (1 ) contains potassium phosphate (30

mM , pH 7.0); DTT (5.0 mM ); EDTA (0.1 mM ); glycerol (50% v/v); and

Triton X-100 (0.1% w/v). React™ 3 buffer was obtained from

Invitrogen (Carlsbad, CA). DNA gel sample solution contains 0.1% (w/v) bromphenol blue; 10% (v/v) glycerol in 0.05 M Tris, pH 7. Ethidium bromide staining solution contains 10 l of a 10 mg/ml solution of ethidium bromide in 100 ml of deionized water. Plasmid DNA—Plasmid DNA (pUC118, Genbank accession no. U07649.1, 3162 bp) [5] is prepared from 250-ml cultures of Esch- erichia coli that have been transformed with pUC118 and grown

as described in Sambrook and Russell [4]. After harvesting by

centrifugation, the plasmid DNA is obtained from the pellet of bacteria by alkaline lysis with SDS and is purified by equilibrium centrifugation in continuous cesium chloride-ethidium bromide gradients. The purified plasmid is suspended in TE buffer to give a final DNA concentration of 1 mg/ml and stored at –20 °C. One 250-ml culture typically yields about 0.5–1 mg of DNA. Plasmid DNA prepared in this way gives consistently better results for this practical that does DNA prepared by resin-based chromato- graphic methods. M13 DNA—A culture of E. coli JM109 is infected with bacte- riophage M13mp18 [6] (Genbank accession no. M77815.1, 7250

29

nt) as described in Sambrook and Russell [4]. The infected cells are added to 250-ml batches of Luria Bertani medium in 2-liter flasks and incubated at 37 °C for 5 h with vigorous aeration. Infected cells and phage particles are separated by centrifuga- tion. Phage particles are precipitated by polyethylene glycol and pelleted by centrifugation. Single-strand circular DNA is isolated from this pellet by the procedures described in Sambrook and Russell [4]. The covalently closed circular form of M13 DNA is isolated from the bacterial pellet by the alkaline lysis procedure also described in Sambrook and Russell [4], and purified by equilibrium centrifugation in cesium chloride-ethidium bromide as described above. Both forms of DNA are suspended at a suitable concentration (0.5–1 mg/ml) in TE buffer and stored at –20 °C. Enzymes—The restriction endonuclease EcoRI and calf thy- mus DNA Topoisomerase I are obtained from Invitrogen. DNA Topoisomerase I is diluted in topoisomerase storage buffer im- mediately prior to the experiment. Agarose Gel Solution—These are made from a 0.7% solution of agarose in TAE buffer.

Sessions 1 and 2

Each group of students is provided with the following:

Horizontal gel box with comb (10 well, session 1; 20 well, session 2). Suitable power supply.

100 ml of 0.7% agarose in 1 TAE buffer. (Melted and held at 55 °C in a water bath. Do not add ethidium bromide to the agarose.)

1 TAE buffer sufficient for gel apparatus used.

100 ml of ethidium bromide staining solution.

1.5-ml microcentrifuge tubes.

Adjustable pipettors (2–20 l, 20 –200 l, 0.1- 1.0 ml) with appropriate tips.

Session 1

Each group of students is provided with the following:

70 l of plasmid DNA ( 0.5–1 mg/ml) in TE buffer.

25 ml of TE buffer.

Spectrophotometer capable of readings at 260 nm.

1-ml UV transparent cuvettes.

Session 2

Each group of students is provided with the following:

20 l of plasmid DNA (200 g/ml in TE buffer).

20 l of M13 CCC DNA (200 g/ml in TE buffer).

20 l of M13 single-stranded circular DNA (200 g/ml in TE buffer).

10 l of calf thymus DNA Topoisomerase I (Invitrogen) (1 unit/ l diluted in topoisomerase dilution buffer immediately prior to the start of the practical).

15 l of topoisomerase reaction buffer (10 ).

5 l of EcoRI (1 unit/ l).

10 l of React™ 3 buffer.

20 l of a mixture of 1 kbp double-stranded DNA size stand- ards (Invitrogen 1 kb ladder).

PROCEDURES

Session 1 and 2

Preparation of Agarose Gels—The 0.7% solution of aga- rose in TAE buffer is prepared and melted prior to the practical period and held at 45 °C in a water bath. Gels with 10 (session 1) or 20 (session 2) sample wells are required. The students pour the gels at the start of each practical, before they begin the rest of the experiment.

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Session 1

Quantification of Plasmid DNA—The students are told the range within which the plasmid DNA concentration is expected to be and are told that double-stranded DNA at

a concentration of 50 g/ml has an absorbance at 260 nm

of 1.0.

After calculating the dilution required to give an absorb- ance of between 0.4 and 0.8 at 260 nm, students prepare

a 1-ml aliquot of this dilution in TE buffer in a 1.5-ml

microcentrifuge tube. After mixing, the absorbance is measured at 260 and 280 nm against a blank of TE. The actual concentration of their plasmid sample is then cal- culated, as is the A 260/280 ratio. Students are asked to find the A 260/280 of highly-purified DNA by referring to the man- uals (Sambrook and Russell [4]) available in the laboratory and to compare their result with this. Preparation of Serially Diluted Plasmid DNA Samples— Each student group prepares a series of dilutions of plas- mid in which 10 l contain the following amount of DNA: 1 g, 500, 200, 100, 50, 20, 10, 5, 2, 1 ng. Dilutions are made into TE buffer. Loading Agarose Gels and Electrophoresis—The combs are removed from the gels and TAE buffer added to just cover the agarose. Ten 2- l drops of loading solution are placed on a strip of parafilm on the bench adjacent to the gel. A 2–20- l pipettor is set to 10 l and used to load the gel, starting with the lowest concentration of DNA. Ten microliters of each sample is mixed with a 2- l drop of loading solution, and the entire 12 l is placed in one of the wells of the agarose gel. This is repeated until all 10 wells have been filled. If the samples are loaded in order from the lowest to highest concentration, there is no need to change the pipette tip between samples. Once all wells are loaded, the lid is placed on the gel apparatus and the leads are connected, checking that the polarity is correct. Stu- dents are encouraged to think about this by asking them which direction they are expecting the deoxyribonucleic acid in their samples to move. The power supply is ad- justed to 100 V. The blue band of bromphenol blue will move about 10 –12 cm in 60 –90 min. After turning off the power, the leads are disconnected and the gel is carefully transferred to a shallow container in which 5 l of a solu- tion of ethidium bromide (10 mg/ml) has been added to 250 ml of water. After staining for 30 – 60 min, the gel is transferred to a container containing water only. The gel is visualized and the results recorded with a suitable UV transilluminator and documentation system. Timing and Student Participation—In this practical, the students take a long time with the calculations and the preparation of the serially diluted plasmid DNA. In a 3-h practical, they manage to get their gels loaded and running but do not usually have time to stain and visualize the gels. This is done for them by a technician, or by the person supervising the laboratory, and the students pick up the record of their experiment the next day.

SESSION 1 RESULTS AND DISCUSSION

The students are told that their plasmid preparation has

a DNA concentration of between 1 and 0.2 mg/ml and that double-stranded DNA at a concentration of 50 g/ml has

BAMBED, Vol. 33, No. 1, pp. 28 –33, 2005

of 50 g/ml has BAMBED, Vol. 33, No. 1, pp. 28 –33, 2005 F IG .

FIG . 1. Amounts of pUC118 plasmid DNA between 1 ng and 1 g were applied to a 0.7% agarose gel in TAE buffer. A potential of 100 V was applied until the bromphenol blue dye band had moved 10 cm from the sample wells. The gel was stained, visualized, and recorded as described under “Materials and Methods.” The amounts of plasmid DNA are shown above the appropriate lanes.

an absorbance at 260 nm (A 260 ) of 1.0. An absorbance of between 0.35 and 0.8 at 260 nm is required for an accurate measurement and this means that a range of dilutions between 1/5 and 1/50 might be appropriate. As they are only provided with a total of 70 l of plasmid they have to make a decision where they balance the potential accu- racy of their measurement with the amount of material they have. Typically a 1/20 dilution to a total of 1 ml will give an A 260 of about 0.4 – 0.8, corresponding to a double-strand DNA concentration of 400 – 800 g/ml for the undiluted sample. The A 260/280 ratio is typically 1.8. The actual DNA concentration is now used for the calculations required to prepare a range of DNA solutions containing between 1 g and 1 ng in 10 l of buffer, and these are used for agarose gel electrophoresis. A typical result for the agarose gel electrophoresis part of the practical is shown in Fig. 1. About 5 ng of DNA in a single band is the limit of detection with ethidium bromide in agarose gels. Depending on the plasmid preparation, the lanes with greater amounts of DNA should show two well-separated bands, and there will often be three bands, particularly if the plasmid DNA preparation has been sub- jected to several freeze-thaw cycles. These results should be discussed with the class as a group at the beginning of the next practical session. The students need to be challenged by asking why a so-called pure preparation of plasmid DNA has more than one band and what the bands might be. They should reach the conclusion that the multiple bands are due to different topoisomers of the plasmid DNA but that there is not sufficient information in the result shown in Fig. 1 to con- clusively identify which of the potential topoisomers are present. This discussion serves as a good introduction to the second part of the practical where they treat plasmid and bacteriophage M13 DNA with a restriction endonucle- ase and a topoisomerase.

Session 2

Treatment of Plasmid and M13 DNA with EcoRI—The reaction mixtures shown in Table I below are set up in microcentrifuge tubes placed on ice. All volumes shown are microliters. Components are added in the order listed, centrifuged briefly, mixed, centrifuged again, and all tubes

TABLE I

Protocol for restriction endonuclease digestion of M13 and plasmid DNA

Tube Number

E1

E2

E3

E4

E5

E6

H 2 O 10 React 3 buffer M13 CCC DNA M13 SS DNA Plasmid DNA

Eco RI restriction endonuclease –

656565

111111

3

3

3

3

––––33

1

1

1

are incubated at 37 °C for 30 min. Ten microliters of DNA gel loading solution is then added to each tube, mixed, and placed on ice. Topoisomerase I Treatment of Plasmid and M13 DNA— Four reaction mixtures are prepared as described in Table II. All volumes are microliters. The tubes are kept on ice and the components added in the order shown. The to- poisomerase is not added until just prior to starting the incubation. Topoisomerase is added to the appropriate tubes, and all tubes are mixed briefly and pulse-spun in the micro- centrifuge. All reaction mixtures are incubated at 37 °C for 30 min. Following the incubation 10 l of gel loading solution is added to each tube, mixed, and the tube placed on ice. Time Course Treatment of Plasmid DNA with Topoi- somerase I—Five microliters of gel loading dye is added to each of six microcentrifuge tubes labeled Topo 0, Topo 1, Topo 2, Topo 5, Topo 10, and Topo 20. All six tubes are placed on ice. The reaction mixture described below is prepared in a single microcentrifuge tube. The components are added in the order listed (Table III) and the tube kept on ice. Before the DNA Topoisomerase I is added, the reaction is mixed well and a 5- l aliquot removed and mixed with the loading dye in the Topo 0 tube. Topoisomerase 1 is added to the remaining reaction mixture. This is mixed well and incubated at 37 °C. At 1, 2 5, 10, and 20 min, a 5- l aliquot is removed and mixed with the loading dye in the appropriately labeled tube and placed on ice. Continue incubating the reaction tube. Electrophoresis of All Samples—After removing the comb from the agarose gel, 1 TAE electrophoresis buffer is added to just cover the agarose. The DNA samples (10 l of each) are placed in the wells of the gel as shown in Fig. 2. After all samples are loaded, the power supply is con- nected and adjusted to 100 V and the gel run until the bromphenol blue dye band has migrated at least 10 cm. The gel is stained with ethidium bromide and visualized as described earlier.

SESSION 2 RESULTS AND DISCUSSION

The results of a typical experiment are shown in Fig. 2. The main reason for failure in this section of the practical is loss of activity of the Topoisomerase I. We have found that it is advisable to obtain a fresh batch of enzyme each year just before the practical is to be run. It is also essential that the enzyme be diluted into topoisomerase storage buffer (not topoisomerase reaction buffer) and that this is done

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T ABLE II

Protocol for DNA Topoisomerase I treatment of M13 DNA

T1

T2

T3

T4

H

10 topoisomerase buffer

M13 CCC DNA M13 SS DNA DNA Topoisomerase I (1/10 dilution in topoisomerase storage buffer)

2 O

4343

1111

5

5

–1–1

5

5

T ABLE III Protocol for DNA Topoisomerase treatment of plasmid DNA

Reaction component

Volume ( l)

H 2 O 10 topoisomerase buffer Plasmid DNA DNA Topoisomerase I

35

5

5

5

buffer Plasmid DNA DNA Topoisomerase I 35 5 5 5 F IG . 2. The reaction

FIG . 2. The reaction products of EcoRI and DNA Topoi- somerase I treatment of M13mp18 and pUC118 DNA were subject to agarose gel electrophoresis as described under “Material and Methods” and the legend to Fig. 1. A 1-kbp ladder (Invitrogen) acted as a size marker for linear duplex DNA molecules.

just before the students require the enzyme. The results obtained in the second part of the experi- ment and shown in Fig. 2 allow the interpretation of the results of Fig. 1 from the first practical session. The EcoRI digest of the plasmid shows that the two (or three) bands of the uncut plasmid give rise to a single band following digestion. The plasmid used was pUC118, which is 3.16 kbp, and the single band resulting from EcoRI digestion has a mobility very close to the 3-kbp band of the marker ladder. If the uncut plasmid DNA shows a third faint band (just evident in lane E5 of Fig. 2), it runs with the same mobility as the product of EcoRI digestion. This allows the students to make the interpretation that the two major bands shown in the uncut plasmid DNA of Figs. 1 and 2 are probably supercoiled and relaxed circular DNA and that the minor band of intermediate mobility is plasmid DNA that has become linearized during plasmid isolation. Two different plasmid preparations have been used to generate the data shown in Figs. 1 and 2. The natural assumption would be that the band with the highest mobility would be the supercoiled plasmid be- cause supercoiling compacts the circular plasmid. This

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32 F IG . 3. Log 1 0 DNA fragment size is plotted against mobility for

FIG . 3. Log 10 DNA fragment size is plotted against mobility for the bands from the 1-kbp ladder shown in Fig. 2 ( ). The mobility of the linear products of EcoRI digestion of pUC118 and M13mp18 have been plotted () and the apparent fragment size calculated.

assumption is supported by the results of the time course of the treatment of the plasmid with DNA Topoisomerase I shown in lanes Topo 0 to Topo 20 in Fig. 2. Here the intensity of the high-mobility band decreases with time of treatment, and there is a corresponding increase in the amount of the slower band. Evident at all time points after zero time are the bands of a ladder of topoisomers, with the distribution moving toward more relaxed forms as digestion proceeds. This result supports the assumption that the faster moving band in the untreated plasmid DNA is highly supercoiled and the slower moving band is re- laxed DNA. The lanes that have the products of EcoRI and Topoi- somerase I treatment of the single-stranded DNA (SS ) of M13 (lanes E3, E4; T1, T2) show no change to the mobility of the DNA band. This suggests that the restriction endo- nuclease and the topoisomerase have no activity toward single-stranded DNA. The effect of EcoRI treatment of the covalently closed double-stranded (ccc) M13 DNA should also be commented on (lanes E1 and E2). Under the con- ditions of the experiment shown, the linear duplex DNA has mobility slightly greater than that of the supercoiled topoisomer in the adjacent lane. This emphasizes the point that it is not possible to assign the topological form to the bands seen following electrophoresis of uncut circular du- plex DNA. In the case of pUC118, linear duplex DNA has mobility between that of the fully supercoiled topoisomer and the relaxed form. The gel shown in Fig. 2 demonstrates a minor anomaly that often occurs with this part of the experiment. The lanes labeled Topo 0 and E5 contain identical DNA, and the relaxed and supercoiled DNA should have the same mobility in each lane. In our hands however, the bands in the Topo 0 lane often have a slightly lower mobility than in the E5 lane. We are not able to explain this anomaly satisfactorily. The absence of the linear 3-kb band in the Topo 0 lane is due to the somewhat lower amount of DNA in this lane. Students should also be required to make a plot of mobility versus log 10 of fragment size for the DNA ladder used. An example derived from the data in Fig. 2 is shown in Fig. 3. Preparing such a plot introduces the concept that, for DNA molecules of the same topological form, mobility is related to the size of the DNA molecule. The

BAMBED, Vol. 33, No. 1, pp. 28 –33, 2005

TABLE IV

Questions for students

1. What physiological manipulations of DNA in vivo require the activity of DNA Topoisomerases?

2. In this experiment the gel electrophoresis was run without ethidium bromide incorporated into the gel. When only linear duplex DNA molecules are being run, it is common practice to include ethidium bromide (2 l of 10 mg/ml per 200 ml) in the agarose solution when the gel is poured. This saves having to stain the gel at the end and also means that the progress of the electrophoresis can be followed during the run. Discuss why the addition of ethidium bromide to the gel could have affected the results of this experiment.

3. The topoisomerase used in this experiment is of type IB. What does this mean in terms of the mechanism of action of the enzyme? In the spread of topoisomers of pUC118, seen in the time course part of the experiment, are the bands separated by one supercoil? If the experiment were repeated with a type IA or a type II topoisomerase, would the result be the same? Explain your answer.

4. In the lane of untreated pUC118 plasmid DNA (lane E5 , Fig. 2), there is a small amount of DNA with a mobility corresponding to the linear duplex DNA generated after digestion by EcoRI ( lane E6 , Fig. 1). How is the DNA in this band generated in the untreated plasmid DNA and what happens to the DNA in this band when the preparation is treated with EcoRI?

DNA fragments in the 1-kb ladder are all linear duplex molecules, and only DNA bands that are also due to linear duplexes may be compared in this way. Students need to decide which of the bands shown in their topoisomerase and EcoRI digests can be used to determine the size of the pUC118 and M13mp18 DNA provided. They should realize the only bands that can be compared in this way are the linear duplex molecules resulting from EcoRI digestion of the double-stranded cir- cular DNA from each DNA in lanes E2 and E6. The mobility plot shown in Fig. 3 shows some deviation from linearity that typically occurs when the gel is run quickly, as is the case here. The mobility of the linear duplex bands of pUC118 and M13mp18 are plotted on the figure, and the corresponding fragment size is shown adjacent to the y -axis. The value for pUC118 (3.13 kbp) is close to the published value of 3.162 kbp. The value for M13mp18 (6.17 kbp) is considerably less than the published value of 7.25 kbp, probably due to the deviation from linearity in this part of the curve. Students can use a graphing program such as Microsoft Excel to prepare the plot of log 10 fragment size versus mobility. We feel, however, that actually preparing and plotting the data is a useful learning exercise. Log/linear graph paper can also be provided, and the use of this is also instructive.

DISCUSSION OF THEORETICAL ASPECTS OF EUKARYOTIC DNA TOPOISOMERASE I ACTIVITY

Students should be encouraged to think about the re- action of type I DNA Topoisomerases, such as the one used in this experiment, by the inclusion of appropriate questions that will require them to consult the references associated with the practical. Examples of these are pro- vided in Table IV. The enzyme used (DNA Topoisomerase I from calf thy- mus) is a eukaryotic topoisomerase and is a type IB en- zyme [3]. Topoisomerases of this class relax both positive and negative supercoils in a reaction that proceeds to

completion and does not require ATP. The type IB topoi- somerases do not share sequence or structural homology with any other topoisomerases and have a different reac- tion mechanism to the prokaryotic type IA enzymes. Type I topoisomerases all change the topology of double- stranded DNA by creating a nick in one of the two strands of the helix and passing the unbroken strand through the gap created, followed by religation. They are all potentially capable of changing the linking number by one, in contrast to the type II topoisomerases that make a double-stranded break and pass duplex DNA through the gap before reli- gation. Consequently type II enzymes change the linking number by 2. Type IB toposiomerases form a covalent intermediate where a tyrosine at the enzyme active site becomes co- valently linked to the 3 -phosphate end of the cleaved strand rather than to the 5 -phosphate end as in the type IA enzymes. An additional point of difference between the type IA and IB topoisomerases is in the nature of the supercoil release by the two classes. Type IA enzymes form a bridge across the broken DNA strand and allow only a single-strand passage across the gap [7]. Type IA enzymes change the linking number by one for each cat- alytic event. In contrast, the mechanism of action of type IB topoisomerases has been proposed to involve a con- trolled rotation of the helix duplex about the uncut DNA strand. This mechanism implies that the linking number can change by greater than one for each breakage and religation event. This has been investigated using the type IB topoisomerase from Vaccinia [8]. In this study, an aver- age change in linking number of five was shown. However, this value can change with the conditions of the experi- ment as shown by Keller [9, 10] using human topisomerase IB and SV40 DNA. This author demonstrated that lowering the reaction temperature to 0 °C increased the number of relaxation intermediates. The results shown in Fig. 2 support this conclusion. Most covalently closed supercoiled DNA has a superheli- cal density of – 0.06 [1]. A plasmid the size of pUC118 should therefore have 18 or 19 superhelical turns. The most obvious intermediate bands shown in the time course of topoisomerase treatment (lanes T0-T20 of Fig. 2) number about 8. This is very similar to the result shown in Fig. 5A of [8] for the relaxation of the related plasmid pUC19 by Vaccinia DNA topoisomerase I, a type IB topoisomerase. Provided that the precautions noted are taken, the suc- cess rate for this practical is high. In the few instances where there has been a total failure of the DNA Topoi- somerase activity, this has always been due either to the

33

use of a batch of enzyme that has been stored for some time, or the dilution of the enzyme too far in advance of when it is required. We find that the first session of the practical gives the students a working knowledge of the sensitivity of ethidium bromide-stained gels to the amount of DNA in a band. It also serves to raise some questions that the students have to consider in the second practical session. The second session illustrates and consolidates their un- derstanding of the activity of restriction endonucleases and DNA Topoisomerases on various topological forms of DNA. In their report on this practical, students need to be encouraged to extract as much information from their data as possible. They should report not only what they ob- serve, but also their interpretation of these observations. For example, rather than merely stating that Topoisomer- ase I does not have any effect on M13 single-strand DNA they should also realize that the concept of topoisomers, as conventionally stated, only applies to duplex DNA mol- ecules that are covalently closed or constrained in some way.

Acknowledgments—Robert Cleaver, Patricia McLenachan, Sherralee Cleland, and Carole Flyger have all made valuable contributions to setting up and maintaining this practical. We also acknowledge the contribution of the students who have at- tempted this practical over the past 10 years and who have provided valuable feedback.

REFERENCES

[1] J. D. Watson, T. A. Baker, S. P. Bell, A. Gann, M. Levine, R. Losick (2004) DNA topology, Molecular Biology of the Gene, 5th Ed., pp. 111–122, Pearson Education, New York. [2] D. Voet, J. G. Voet (2004) Supercoiled DNA, Biochemistry, 3rd Ed., pp. 1122–1133, John Wiley & Sons, New York. [3] J. J. Champoux (2001) DNA topoisomerases: Structure, function, and mechanism, Annu. Rev. Biochem. 70 , 369 – 413. [4] J. Sambrook, D. W. Russell (2001) Molecular Cloning: A Laboratory Manual , 3rd Ed., Cold Spring Harbor Laboratory Press, New York. [5] J. Viera, J. Messing (1987) Production of single-stranded plasmid DNA, Methods in Enzymology (R. Wu, L. Grossman, eds.) Vol. 153, pp. 1–11, Academic Press, San Diego, CA. [6] C. Yanisch-Perron, J. Vieira, J. Messing (1985) Improved M13 phage cloning vectors and host strains: Nucleotide sequences of the M13mp18 and pUC19 vectors, Gene 33 , 103–119. [7] J. J. Champoux (1990) Mechanistic aspects of type-I topoisomer- ases, DNA Topology and Its Biological Effects (N. R. Cozzarelli, J. C. Wang, eds.) pp. 217–242, Cold Spring Harbor Laboratory Press, Plainview, NY. [8] J. Stivers, T. Harris, A. Mildvan (1997) Vaccinia DNA topoisomerase I:

Evidence supporting a free rotation mechanism for DNA supercoil relaxation, Biochemistry 36 , 5212–5222. [9] W. Keller (1975) Characterization of purified DNA-relaxing enzyme

from human tissue culture cells, Proc. Natl. Acad. Sci. U. S. A. 72, 2550 –2554. [10] W. Keller (1975) Determination of the number of superhelical turns in simian virus 40 DNA by gel electrophoresis, Proc. Natl. Acad. Sci.

U. S. A. 72 , 4876 – 4880.