Analytical Biochemistry 425 (2012) 76–79

Contents lists available at SciVerse ScienceDirect

Analytical Biochemistry

journal homepage: www.elsevier .com/locate /yabio

Notes & Tips

On-chip microelectrophoresis for the study of in vitro nonhomologousend-joining DNA double-strand break repair

Catherine Charles a,b, Moustapha Ouedraogo c, Alexandra Belayew d, Pierre Duez a,b,⇑a Department of Therapeutic Chemistry and Pharmacognosy, Université de Mons, 7000 Mons, Belgiumb Laboratory of Pharmacognosy, Bromatology and Human Nutrition, Université Libre de Bruxelles, B-1050 Brussels, Belgiumc Laboratory of Pharmacology and Toxicology, Health Sciences Faculty, University of Ouagadougou, 03 BP 7021 Ouagadougou 03, Burkina Fasod Laboratory of Molecular Biology, Université de Mons, 7000 Mons, Belgium

a r t i c l e i n f o

Article history:Received 28 February 2012Received in revised form 3 March 2012Accepted 5 March 2012Available online 10 March 2012

Keywords:ChemopreventionFlavonoidsDNA repairMicroelectrophoresis

0003-2697/$ - see front matter � 2012 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.ab.2012.03.003

⇑ Corresponding author at: Department of Therapecognosy, Université de Mons, 7000 Mons, Belgium.

E-mail address: pduez@ulb.ac.be (P. Duez).1 Abbreviations used: DSB, double-strand break; HR

NHEJ, nonhomologous end joining; DNA–PKcs, DNcatalytic subunit; XRCC4, X-ray cross-complementavariance.

a b s t r a c t

Oligomerization of linearized plasmids by nuclear proteins extracts, a recognized measure of nonhomol-ogous end-joining (NHEJ) repair capacity, is typically assessed through agarose gel electrophoresis, alabor-intensive procedure. In the current study, a more convenient NHEJ assay was developed usingmicrochips that allow scaled-down separation and quantification. This microchip method allows a con-siderable reduction in sample amount and analysis time with similar costs and comparable or slightlybetter precision. Data obtained with quercetin and wortmannin show that the method can be appliedto the screening of food components and natural products for positive and negative modulators of NHEJ,potential chemopreventive and indirect genotoxic compounds, respectively.

� 2012 Elsevier Inc. All rights reserved.

DNA double-strand breaks (DSBs),1 the most critical lesions interms of cell death, result from endogenous and/or exogenous pro-cesses. Consequences of misrepaired DSBs include genomic instabil-ity, aging, cancer development, and cell death. Therefore, efficientDSB repair is critical for cell integrity. The repair of DSBs involvestwo main different pathways, homologous recombination (HR) andnonhomologous end joining (NHEJ), which was the focus of the cur-rent study [1,2]. NHEJ joins two broken ends directly end to end,does not require a homologous DNA template, and so can operatewhen only one chromosomal copy is available. Thus, NHEJ is a majorDSB repair mechanism in higher eukaryotes during the G1/early Sphase of the cell cycle, whereas HR is predominant in the late S/G2phase when a sister chromatid is available [3]. The NHEJ pathway re-quires the concerted action of several components, including threecore factors: (i) the DNA-end-binding Ku70/Ku80 heterodimer, (ii)the DNA-dependent protein kinase catalytic subunit (DNA–PKcs)that forms a complex with the Artemis (DNA–PKcs/Artemis) nucle-ase, and (iii) the X-ray cross-complementation 4 (XRCC4)/ligase IVcomplex. The XRCC4-like factor/Cernunnos (XLF/Cer), the nucleaseMre11, the polymerases (Pols k, l, and terminal deoxynucleotidyl

ll rights reserved.

utic Chemistry and Pharma-

, homologous recombination;A-dependent protein kinasetion 4; ANOVA, analysis of

transferase), tyrosyl-DNA–phosphodiesterase 1, and exonuclease 1can also be involved. The precise manner in which these factorsare employed depends on the nature of the DNA termini [4,5]. Giventhe high complexity of this pathway and the number of proteins andcofactors involved, it is likely that xenobiotics, including naturalcompounds, may interfere in the process, activating or inhibiting re-pair [6].

The current work aimed to increase the throughput of in vitroNHEJ assays for screening purposes and to study the potential ofquercetin, a well-known natural flavonol particularly abundant inour diet, to modulate in vitro DSB repair by the NHEJ pathway.

Typical in vitro NHEJ assays are based on the oligomerization oflinearized plasmids by cellular or nuclear protein extracts followedby the analysis of the reaction products on agarose gel electropho-resis [1,4]. However, this manual method is time-consuming, andits resolution, reproducibility, and detection limits permit at besta semiquantification of DNA bands [7]. Microchip electrophoresisprovides an attractive alternative with shorter analysis time andlower reagent and sample consumption, which is particularly suit-able for screening a large number of compounds [7]. Although this‘‘lab-on-a-chip’’ technique has been successfully used for variouspurposes [8], its application to NHEJ assays was not granted dueto the complexity of the reaction mixtures (total protein extracts,cofactors, and salts), the large size of the plasmid oligomers, andthe low DNA concentration.

The LHCN-M2 human myoblast cell line, immortalized by trans-duction with hTERT and retroviral expression vectors to overcome

Notes & Tips / Anal. Biochem. 425 (2012) 76–79 77

senescence [9], was selected for this study instead of cancer celllines with uncertain repair capacities. The plasmid pGEM-7Z(3 kB), linearized by the restriction enzyme Cfr9I (GGCC cohesiveends), was incubated with a nuclear extract obtained from eithercontrol or quercetin-pretreated cells; quercetin was added to theculture medium at a nonlethal concentration (30 lM; microculturetetrazolium test over 24 h) and left in contact with nonconfluentcells for 24 h before the realization of nuclear extract (detailedmaterials and methods are available in the Supplementary mate-rial). The proteins from the extract ‘‘repair’’ the linearized plasmidsby end-to-end joining, resulting in the formation of plasmid oligo-mers. These reaction products were separated and quantified bothby agarose gel electrophoresis, followed by the treatment of gelpictures with VideoScan CAMAG software, and by microchip elec-trophoresis (Experion Automated Electrophoresis System, 12KDNA chips) (Fig. 1). Microchip analyses were at first quite unsuc-cessful due to microchannel clogging that was prevented by exten-sive precentrifugation (16,000 g for 15 min) of test solutions. TheDNA oligomers were suitably resolved on microchip with a highspeed and an excellent signal-to-noise ratio, allowing efficientintegration of the peaks. For both methods, the multimer propor-tions regularly increase with protein concentration up to 0.5 lg/ll (Fig. 2A). Higher protein concentrations result in plasmid frag-mentation, probably due to aspecific endonuclease activities,reducing both monomer and multimer signals and causing smears.Such results are exemplified for the 1 lg/ll concentration (Fig. 2A).Apart for the experiments with this excessive protein concentra-tion, no significant difference was observed between on-gel andon-chip results. Total precision, computed by two-way analysis

Fig.1. Comparison of agarose gel electrophoresis (A) and microchip electrophoresis (B). Plower molecular weight marker; (UM) upper molecular weight marker.

of variance (ANOVA; 3 biological replicates analyzed on 3 differentdays) for both methods, indicates a slightly better precision for theon-chip method (Fig. 2B).

A well-known inhibitor of phosphatidylinositol-3-kinase, wort-mannin, was tested as a positive control for NHEJ inhibition. Wort-mannin (20 lM; 10 min pretreatment of nuclear extract; detailedmaterials and methods are available in the Supplementary mate-rial) caused an inhibition of approximately 12% on both methods(gel/multimer ratio = 12.1 ± 3.2%, n = 3; microchip/multimer ra-tio = 12.3 ± 1.9%, n = 3); this modest effect is in line with a previousstudy showing that wortmannin moderately reduces the NHEJactivity of nuclear protein extracts [10]. On the immortalized hu-man myoblast model, quercetin increased plasmid oligomeriza-tion, indicating a positive modulation of NHEJ (Fig. 2C). Becausethis on-chip model is able to detect both a decrease and an increasein repair activity, it is amenable to screening for potential NHEJinterfering compounds.

Quercetin, a flavonol largely investigated for multitargeted anti-mutagenic, antiproliferative, and antioxidant activities, plays a rolein the regulation of cell signaling, cell cycle, and apoptosis. The cur-rent study shows that this flavonoid, at relevant physiological con-centrations, exhibits a stimulatory effect on the NHEJ mechanismsthat could represent an original cancer chemopreventionmechanism.

Defects in either the NHEJ or HR pathway are known to causegenome instability and promote tumorigenesis [2]. Although NHEJis often considered as an ‘‘error-prone’’ pathway whereas HR is de-scribed as ‘‘error free’’ [2], both of these pathways can in fact beprecise or imprecise, depending on a series of factors [11]. The

eaks: (1) plasmid monomer; (2) dimer; (3) trimer; (4) tetramer; (5) pentamer; (LM)

A

0.25

0.38

0.50

1.00

0

20

40

60Gels

Chips

NS

NS

NS

***

Nuclear protein concentration (µg/µl)

% M

ultim

ers

B

(a) RSD = relative standard deviation; 3 experiments in triplicate. (b) Summation of multimer areas (up to 4 units).

C Agarose gels

0.25

0.38

0.50

1.00

0

50

100

150

200

250

*

NS*

*

Nuclear protein concentration (µg/µl)

% M

ultim

ers

Microchips

0.25

0.38

0.50

1.00

0

50

100

150

200

250 ***

****

NS

Nuclear protein concentration (µg/µl)

% M

ultim

ers

Control

Quercetin

Control

Quercetin

Fig.2. (A) Influence of nuclear protein concentration. A comparison between agarose gel electrophoresis and automated microchip electrophoresis methods is shown, withproportions of multimers as a function of nuclear protein concentration (reaction time = 20 min; three experiments in triplicate; means and standard deviations). An ANOVA-protected Student’s t test for the difference between gels and chips was performed (NS, p > 0.05; ⁄⁄⁄p < 0.001). (B) Precision of agarose gel electrophoresis and automatedmicrochip electrophoresis methods as function of nuclear protein concentration and polymerization degree of plasmids. (C) Influence of quercetin on DNA repair. Effects ofquercetin on plasmid oligomerization were measured by agarose gel electrophoresis (A) and microchip electrophoresis (B) methods. Proportions of multimers as a function ofnuclear protein concentration are shown (reaction time = 20 min; three experiments in triplicate; means and standard deviations; data were normalized to their respectivecontrol). An ANOVA-protected Student’s t test for the difference between gels and chips was performed (NS, p > 0.05; ⁄p < 0.05; ⁄⁄p < 0.01; ⁄⁄⁄p < 0.001).

78 Notes & Tips / Anal. Biochem. 425 (2012) 76–79

components required by the classical NHEJ (DNA–PK complex,XRCC4/DNA ligase IV, Artemis, and BRCA1 gene) act as caretakersof the mammalian genome [12]; therefore, NHEJ-deficient cellsshow higher rates of genome rearrangements [13]. In large repet-itive genomes of plants and animals, a too efficient HR may leadto deleterious genomic rearrangements; thus, NHEJ may be a saferchoice. Cells seem to favor the accumulation of small mutationsresulting from NHEJ instead of chromosomal translocation or dele-tion caused by HR if the choice of the homology partner is inappro-priate [14]. Activation of NHEJ may effectively participate in thestability of the genome.

To the best of our knowledge, this work is the first application ofthe microchip electrophoresis technology to assess DNA repairmodulation by natural compounds in a highly complex reactionmixture. Compared with agarose gels, microchips allow automa-tion, replacing all of the electrophoresis steps (separation, staining,destaining, band detection, and data analysis), and strongly reducethe manual and time-consuming procedures associated with gelhandling. Although the precision of the results is only slightly bet-ter on the microchips, the reproducibility of casting, loading, andrunning agarose gels required dexterity, and integrating gel elec-tropherograms is much more difficult and time-consuming. Aga-rose gel performances are indeed influenced by gel and bufferpreparation, loaded DNA amount, and manual procedures [8].Microchip miniaturization brings additional advantages. On theone hand, the reduction of reagent and sample consumption isconsiderable. Due to the high sensitivity of laser-induced fluores-cence detection, microchip NHEJ experiments required only1.1 ll of DNA sample (11 ng of DNA), whereas agarose gel electro-phoresis implied at least 15 ll (170 ng of DNA). On the other hand,microchip electrophoresis allows a simplification of analyses withhigh efficiency and sensibly increased throughput (analysis of 11samples in 30 min for the system we use). Finally, the exposureto hazardous chemicals is reduced, and single-use microchips gen-erate low waste [8].

Microchip electrophoresis is a promising tool to study in vitrothe potential of naturally occurring compounds to modulate the re-pair of DNA DSBs through the NHEJ pathway. Depending on restric-tion enzymes used, different types of lesions (e.g., cohesive,noncohesive, blunted) can be induced, widening the scope of themethod. Because defects in DNA DSB repair are allegedly essentialfor carcinogenesis [12], NHEJ modulation could be indirect geno-toxicity enhancement or avoidance mechanisms worthy ofscreening.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ab.2012.03.003.

References

[1] G. Iliakis, B. Rosidi, M. Wang, H. Wang, Plasmid-based assays for DNA end-joining in vitro, Methods Mol. Biol. 314 (2006) 123–131.

[2] M. Shrivastav, L.P. De Haro, J.A. Nickoloff, Regulation of DNA double-strandbreak repair pathway choice, Cell Res. 18 (2008) 134–147.

[3] K. Rothkamm, I. Kruger, L.H. Thompson, M. Lobrich, Pathways of DNA double-strand break repair during the mammalian cell cycle, Mol. Cell Biol. 23 (2003)5706–5715.

[4] E. Pastwa, R.I. Somiari, M. Malinowski, S.B. Somiari, T.A. Winters, In vitro non-homologous DNA end joining assays––the 20th anniversary, Int. J. Biochem.Cell Biol. 41 (2009) 1254–1260.

[5] K. Bahmed, A. Seth, K.C. Nitiss, J.L. Nitiss, End-processing during non-homologous end-joining: a role for exonuclease 1, Nucleic Acids Res. 39(2011) 970–978.

[6] H. Bernstein, C. Crowley-Skillicorn, C. Bernstein, C.M. Payne, K. Dvorak, H.Garewal, Dietary compounds that enhance DNA repair and their relevance tocancer and aging, in: B.R. Landseer (Ed.), New Research on DNA Repair, NovaScience, New York, 2007, pp. 99–113.

[7] L. Ding, K. Williams, W. Ausserer, L. Bousse, R. Dubrow, Analysis of plasmidsamples on a microchip, Anal. Biochem. 316 (2003) 92–102.

[8] V. Dolnik, S. Liu, Applications of capillary electrophoresis on microchip, J. Sep.Sci. 28 (2005) 1994–2009.

[9] C.H. Zhu, V. Mouly, R.N. Cooper, K. Mamchaoui, A. Bigot, J.W. Shay, J.P. Di Santo,G.S. Butler-Browne, W.E. Wright, Cellular senescence in human myoblasts is

Notes & Tips / Anal. Biochem. 425 (2012) 76–79 79

overcome by human telomerase reverse transcriptase and cyclin-dependentkinase 4: Consequences in aging muscle and therapeutic strategies formuscular dystrophies, Aging Cell 6 (2007) 515–523.

[10] R. Perrault, H. Wang, M. Wang, B. Rosidi, G. Iliakis, Backup pathways of NHEJare suppressed by DNA–PK, J. Cell Biochem. 92 (2004) 781–794.

[11] S.T. Durant, J.A. Nickoloff, Good timing in the cell cycle for precise DNA repairby BRCA1, Cell Cycle 4 (2005) 1216–1222.

[12] S. Burma, B.P. Chen, D.J. Chen, Role of non-homologous end joining (NHEJ) inmaintaining genomic integrity, DNA Repair (Amst.) 5 (2006) 1042–1048.

[13] L. Deriano, H. Merle-Beral, O. Guipaud, L. Sabatier, J. Delic, Mutagenicity ofnon-homologous end joining DNA repair in a resistant subset of humanchronic lymphocytic leukaemia B cells, Br. J. Haematol. 133 (2006) 520–525.

[14] M.R. Lieber, Z.E. Karanjawala, Ageing, repetitive genomes and DNA damage,Nat. Rev. Mol. Cell Biol. 5 (2004) 69–75.