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Lotte Bjergbæk (ed.), DNA Repair Protocols, Methods in Molecular Biology, vol. 920,DOI 10.1007/978-1-61779-998-3_22, © Springer Science+Business Media New York 2012

Chapter 22

DNA Double-Strand Break Damage and Repair Assessed by Pulsed-Field Gel Electrophoresis

Helen E. Bryant

Abstract

Pulsed- fi eld gel electrophoresis (PFGE) is a technique for resolving large DNA molecules (up to 10 Mb). Using multiple pairs of electrodes DNA is subject to an alternating electric fi eld through a solid agarose matrix. As the current changes direction the reorientation time of DNA is proportional to molecular weight; thus fragments are separated in the gel based on their size. Here we describe the use of PFGE to analyze DNA double-strand break formation and repair in human chromosomal DNA.

Key words: Pulsed- fi eld gel electrophoresis , PFGE , DNA double-strand breaks , Genotoxic , Apoptosis , DNA damage , DNA repair , Method , Protocol

In conventional gel electrophoresis a constant current is used to sieve small molecules of DNA through a slab of agarose. However as large pieces of DNA exist as randomly coiled molecules they need signi fi cant unraveling before they can pass into and weave their way through the gel. Such unraveling and weaving (also known as reptation) means that passage through the gel is no longer proportional to molecular weight. The practical resolution limit for conventional agarose gel electrophoresis is therefore about 50 kb. To overcome this limit Schwartz and Cantor developed pulsed- fi eld gel electrophoresis (PFGE) ( 1 ) . Cells are embedded in agarose plugs, then subject to proteinase K digestion leaving the DNA intact in the plug. After washing away the protein, plugs are set into an agarose gel prior to PFGE. During electrophoresis the reptation of the DNA molecules through a matrix is exploited; the agarose gel is subject to a homogenous electric fi eld ( 2 ) that alternates in direction; as the direction alters the DNA molecules reorientate

1. Introduction

316 H.E. Bryant

to the new direction, maximum mobility only occurring when molecules are fully aligned with the fi eld. The time taken to reori-entate is proportional to DNA molecular weight; however once reorientation has occurred, migration through the gel in a size-dependent manner is lost. The frequency of switching between fi elds is therefore set to optimize the time spent undergoing reori-entation considering the resolution required; here an initial switch time of 60 s is used; this increases to 240 s over 24 h. The result is that DNA zig-zags across the gel and that net electrophoretic mobility is dependent on size. To achieve optimal separation an obtuse reorientation angle is required; 120° is usually considered adequate ( 3 ) and is used in this protocol. After electrophoresis DNA is visualized using ethidium bromide. Here we describe how double-strand breaks induced by endogenous or exogenous agents can be visualized ( 4 ) and in a variation on this how repair of such breaks over time can also be seen ( 5, 6 ) .

1. Phosphate-buffered saline: Dissolve 8 g of NaCl, 0.2 g KCl, 1.44 g Na 2 HPO 4 , and 0.24 g KH 2 PO 4 in 800 ml distilled H 2 O. Adjust pH to 7.4, adjust volume to 1 L with additional dis-tilled H 2 O, and sterilize by autoclaving. Store at room temperature.

2. 0.5 M EDTA: Dissolve 186.1 g Na 2 EDTA·2H 2 O in 800 ml dis-tilled water. pH to 8.0 with NaOH (~20 g of NaOH pellets). EDTA will dissolve at pH 8.0. Adjust volume to 1 L with distilled water. Sterilize by autoclaving and store at room temperature.

3. Proteinase K solution: Dissolve 100 mg N -laurylsarcosyl and 10 mg Proteinase K into 10 ml 0.5 M EDTA. Scale up as required (1 ml/plug) and store at room temperature.

4. 10× TBE: Dissolve 108 g Tris base and 55 g Boric acid in 800 ml distilled H 2 O, add 40 ml 0.5 M EDTA (pH 8.0), adjust volume to 1 L, sterilize by autoclaving, and store at room temperature.

5. 0.5× TBE: Dilute 250 ml 10× TBE into 4.75 L distilled water. 6. TE10: Dissolve 1.21 g Tris base in 900 ml distilled water, add

20 ml 0.5 M EDTA, adjust volume to 1 L, sterilize by auto-claving, and prechill in fridge before use.

7. TE100: Dissolve 1.21 g Tris base in 700 ml distilled water, add 200 ml 0.5 M EDTA, adjust volume to 1 L, sterilize by auto-claving, and prechill in fridge before use.

8. RNAse A solution: Dissolve 1 mg RNAse powder in 10 ml distilled water, aliquot into 1 ml lots, and store at −20 °C.

2. Materials

2.1. Buffers

31722 DNA Double-Strand Break Damage and Repair Assessed…

9. Staining solution: Add 25 m l 10 mg/ml stock ethidium bromide to 500 ml 0.5× TBE.

10. Marker: Commercially available CHEF DNA size marker of Saccharomyces cerevisiae chromosomes comes in agarose blocks that can be cut into plugs.

11. Preparation of agarose for plugs: Use InCert ® Agarose, a low gelling ( £ 30°C) and melting ( £ 70°C) temperature, high-purity agarose. Before starting set a water bath at 50–60 °C. Weigh out 0.15 g agarose powder, add 10 ml room temperature PBS to a Falcon tube, add the agarose powder to the PBS while gently agitating, weigh the tube containing PBS and agarose and note weight, loosen lid on tube, heat in a microwave oven with the power setting on medium using several short (10 s) intervals with gentle swirling in between heating to resuspend the powder, continue till there are no visible particles (see Note 1). Reweigh tube, add warm distilled water to obtain the initial weight, and mix. Place tube in water bath until needed.

12. Preparation of electrophoresis gel: Use Chromosomal grade agarose, dissolve 2.8 g agarose in 350 ml 0.5 %TBE, heat on medium setting in microwave for 5 min or until agarose is com-pletely dissolved. Allow to cool till comfortable to hold in hands, swirling periodically to prevent the gel from setting. Once at an appropriate temp, add a stir bar and transfer to heated stir block set at around 40 °C. For pouring gel see method below.

In addition to standard lab equipment you need,

1. Water bath set at 50–60 °C. 2. Microwave oven. 3. Refrigerated centrifuge to spin 15 ml Falcon tubes at 200 × g . 4. The protocol below has been optimized for use with the

BioRad CHEFIII system; other commercial systems are avail-able or a system can be homemade ( 7 ) . Items 5– 7 below are accessories to the CHEFIII system.

5. Disposable PFGE plug molds (see Note 2). 6. Standard PFGE casting stand and comb. 7. Screened cap to fi t a 50 ml Falcon tube.

1. Seed cells at 1–4 × 10 6 /100 mm dish (see Note 3). 2. Leave 4 h to attach. 3. Treat cells with double-strand break-inducing agent as required

(see Notes 4 and 5 ).

2.2. Equipment

3. Methods

3.1. Treatment of Cells

318 H.E. Bryant

1. Before starting make up 1.5 % InCert agarose and proteinase K buffer (see above), precool the centrifuge, and collect ice. Also label (see Note 6) and prepare the plug molds by ensuring that they are clean and that the bottom is well stuck.

2. Taking a maximum of four plates at a time, trypsinize cells gently off the plate (see Note 7).

3. Resuspend cells in 10 ml media and place on ice. 4. Count harvested cells. 5. Pellet 1 × 10 6 cells/plug by centrifuging the appropriate vol-

ume at 4 °C for 3 min at 200 × g in a 15 ml Falcon tube. 6. Remove supernate from cells. 7. Gently resuspend each pellet in 50 m l PBS. 8. To one tube at a time, add 50 m l warm Incert agarose, gently

resuspend, and then immediately pipette 80 m l into the plug mold (see Notes 8 and 9 ).

9. Once you have made all the plugs, leave at 4 °C for 10 min to set. 10. Meanwhile add 1 ml/plug of proteinase K buffer to each of

the appropriate number of 50 ml Falcon tubes (see Note 10). 11. Using the plastic insert provided push plug out of mold into

proteinase K buffer and incubate at 50 °C for 48 h.

1. Wash plugs 2× 30 min in cold TE10 (see Note 11). 2. Add 100 m l 1 mg/ml RNAseA to each tube and incubate at

37 °C for 1 h. 3. Wash 1× 30 min TE10. 4. Wash 1× 30 min TE100 (see Note 12). 5. Store in TE100 at 4 °C (see Note 13).

1. Rinse PFGE tank out with distilled water, then add 0.5× TBE buffer, turn on pump, and set chiller to 14 °C (Fig. 1 ) (see Note 14).

2. Prepare electrophoresis gel (see above). 3. While the agarose is cooling, place each plug on a tooth of the

comb (Fig. 2 ) (see Notes 15– 17 ) and allow to dry a little (see Note 18).

4. Position the comb (with plugs on) in the casting stand (Fig. 3 ), pour gel around to ¾ depth of the plugs, and allow gel to set (see Note 19). Remove comb and top up gel so that plugs are covered; the plugs should stay in place in the gel. Allow the rest of gel to set.

3.2. Preparation of Agarose Plugs

3.3. Washing Plugs

3.4. Electrophoresis

31922 DNA Double-Strand Break Damage and Repair Assessed…

5. Transfer gel to electrophoresis tank; for the CHEFIII system the settings are

PBS protocol Block 1

Initial switch time 60 s

Final switch time 240 s

Run time 24 h

Volts/cm 4 V

Included angle 120

6. After electrophoresis transfer the gel to staining solution over-night. Visualize on UV light box (see Note 20).

electrodes

tank

Gel with plugsOn back plate

Gel positioning plate

ChillerUnit

Control box

tubing

Pump

Electricalconnection

Temperatureprobe

Safetycable

Fig. 1. BioRAD CHEFIII system setup.

PLUG COMB

Fig. 2. From above ; comb with plugs laid on top. The comb should be placed on the bench top; plugs are then lifted from the tube onto the teeth of the comb using a small spatula. Tissue is used to blot away excess buffer and plugs left on the comb for around 30 min, until plugs do not slip off teeth when lifted.

320 H.E. Bryant

As above BUT treat enough cells to make multiple plugs, then at step 7 in Subheading 3.2 resuspend pellet in warm medium and not PBS, and at step 8 in Subheading 3.2 resuspend in medium/incert agarose rather than PSB/incert agarose using the same ratios. Then after step 9 in Subheading 3.2 incubate each plug at 37 °C in 1 ml/plug medium in a 50 ml Falcon tube for desired repair time. Finally wash plugs with PBS before transferring each plug to a fresh 50 ml tube containing 1 ml/plug proteinase K buf-fer and continuing with the protocol at step 11 in Subheading 3.2 . Alternately treat cells with damaging agent on the 100 mm dish, then wash off with PBS, replace with normal medium, and allow to repair on the dish prior to making plugs in the standard way.

1. When the agarose comes to a boil, handle solution very care-fully. Allow to stand brie fl y at room temperature before releas-ing the air by gentle swirling.

2. Although PFGE molds are sold as disposable molds, you can wash them out and reuse them using a roll of thin electrical tape.

3. Seed cells so that after treatment at least 1 × 10 6 cells remain. 4. Remember to include a no treatment control. 5. The limit for detection is approximately the number of DSBs

produced by 5 Gy ionizing radiation. 6. Use a non-permanent marker if you want to reuse the molds. 7. Do not bang the plates; this can induce DSBs.

3.5. Repair Assay

4. Notes

COMB

PLUG

CASTING STAND

BACKPLATE

Empty wells

a b

Fig. 3. From above ; ( a ) Casting unit with backplate and comb/plugs in place, ( b ) with comb removed. The backplate is positioned in the casting unit; the comb with plugs adhered to it is then carefully lowered into the casting unit ( a ). Warm agarose is then poured up to ¾ of plug depth. Once this has set the comb is carefully removed leaving the plugs in place ( b ); fi nally warm agarose is poured over the plugs, fi lling the wells left by the comb and covering the plugs.

32122 DNA Double-Strand Break Damage and Repair Assessed…

8. Pipette gently without introducing any bubbles. 9. To fi ll the mold touch pipette to the inside edge of the mold

and gently push the agarose in so that it slides down the edge. 10. Duplicate or triplicate plugs can go in the same tube with 2 ml

or 3 ml of proteinase k buffer, respectively. 11. To wash remove the normal Falcon tube lid and replace with the

screened cap. The screened cap screws onto a 50 ml Falcon tube; it contains holes that allow you to pour away liquid without los-ing the plug; you can then pour the next wash solution into the tube through the screened cap; fi nally remove the screened cap and replace with normal lid for the duration of the wash.

12. After washing the plugs should become clear. 13. Samples are stable for at least 1 month at 4 °C. 14. If the buffer is not suf fi ciently chilled the gel will not run prop-

erly and a series of smudged bands will be seen. A common error is to turn on the chiller without the pump; when you remember to turn on the pump buffer will not circulate because buffer has frozen in the chiller. Turn off chiller, wait for 30 min, and then try turning on pump again.

15. Include marker plug, and cut a section from the slab supplied. 16. A small spatula is useful to transfer plugs to the comb. 17. Blot away excess liquid using tissue. 18. To check if dry enough gently tilt the comb; the plugs should

stay on the comb; if they begin to slide return comb to hori-zontal and wait longer. This takes about 20 min.

19. Return the remaining agarose to the stir block. 20. This can be improved by Southern blotting and hybridization

to radiolabeled fragmented total genomic DNA probe.

References

1. Schwartz DC, Cantor CR (1984) Separation of yeast chromosome-sized DNAs by pulsed fi eld gradient gel electrophoresis. Cell 37:67–75

2. Gardiner K, Laas W, Patterson D (1986) Fractionation of large mammalian DNA restric-tion fragments using vertical pulsed- fi eld gradi-ent gel electrophoresis. Somat Cell Mol Genet 12:185–195

3. Chu G, Vollrath D, Davis RW (1986) Separation of large DNA molecules by contour-clamped homogeneous electric fi elds. Science 234:1582–1585

4. Saleh-Gohari N, Bryant HE, Schultz N, Parker KM, Cassel TN, Helleday T (2005) Spontaneous homologous recombination is induced by

collapsed replication forks that are caused by endogenous DNA single-strand breaks. Mol Cell Biol 25:7158–7169

5. Bryant HE, Ying S, Helleday T (2006) Homologous recombination is involved in repair of chromium-induced DNA damage in mammalian cells. Mutat Res 599:116–123

6. Ying S, Myers K, Bottomley S, Helleday T, Bryant HE (2009) BRCA2-dependent homol-ogous recombination is required for repair of Arsenite-induced replication lesions in mam-malian cells. Nucleic Acids Res 37:5105–5113

7. Herschleb J, Ananiev G, Schwartz DC (2007) Pulsed- fi eld gel electrophoresis. Nat Protoc 2:677–684