Research Article Configuration Transitions of Free Circular DNA System Induced by Nicks

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1 Nanomaterial Volume 215, Article ID , 7 page Reearch Article Configuration Tranition of Free Circular DNA Sytem Induced by Chao Ji, 1,2 Lingyun Zhang, 1 and Pengye Wang 1 1 Beijing National Laboratory for Condened Matter Phyic, Intitute of Phyic, Chinee Academy of Science, Beijing 119, China 2 College of Biological Science, Weifang Medical Univerity, Shandong 26153, China Correpondence hould be addreed to Lingyun Zhang; lyzhang@iphy.ac.cn and Pengye Wang; pywang@iphy.ac.cn Received 27 May 215; Accepted 7 July 215 Academic Editor: Song Cui Copyright 215 Chao Ji et al. Thi i an open acce article ditributed under the Creative Common Attribution Licene, which permit unretricted ue, ditribution, and reproduction in any medium, provided the original work i properly cited. have important function in the biological function of DNA-mediated ytem. However, the configuration tranition of DNA molecule induced by the preence of nick have not been quantitatively invetigated. Thi tudy aim to analyze the configuration tranition of free circular DNA ytem induced by nick. Uing atomic force microcopy, two configuration tate were oberved in the free circular DNA ytem with different nick number. To undertand the tranmiion of torional energy among DNA bae pair, we defined the effective length and nicking angle. In the free DNA ytem, a torional energy of 233 bp can be completely releaed by nick. Baed on the experimental and quantitative reult, we propoe a phyical mechanim to explain the configuration tranition of the free circular DNA ytem induced by nick. Thi tudy and the preented method are very ueful in undertanding the phyical mechanim of nick in DNA-mediated ytem. 1. Introduction A nick i a dicontinuity in the double helical tructure of DNA. It i a ingle-trand break in which a phophodieter bond between adjacent nucleotide i cleaved [1]. ed DNA ha an important function in the biological function of cell procee. Topoiomerae I of the DNA recombination ytem create tranient DNA break via a covalent bond betweenthecleavedtrandandtheenzyme;thibreaki generally initiated by nick on bae pair (bp) [2, 3]. ed DNAhaalobeenuedinthetudyofthebaeexciion repair pathway, which i the primary repair ytem ued to maintain the genome integrity in mammalian cell [4 7]. In addition, double nicking i applied to enhance genome editing pecificity in targeted genome editing technologie [8 1]. And the crytal tructure of polymerae β complex with nicked DNA reveal that polymerae β bind nicked DNA with a 9 kink [11]. Thi finding demontrate that nicked DNA i more flexible than double-tranded DNA. Thu, nicked DNA i more prone to change in configuration. Protein can be timulated by nick to bind to DNA and carry out their pecific biological function [12]. However, configuration tranition of DNA molecule induced by the preence of nick have yet to be quantitatively invetigated. It invetigation will be very helpful in undertanding the important function of the DNA configuration tranition. In thi tudy, we preent a method to analyze quantitatively the configuration tranition of circular DNA induced by nick uing atomic force microcopy (AFM). We tudied the configuration tate of circular DNA containing differentnumberofnick.weobervedtwo(flatcircularand interecting tate) configuration tate in the circular DNA ytem. Further, for revealing the phyical property of nick in DNA molecule, we defined the effective length, releae length, and nicking angle in topological analyi to tudy the tranmiion of torional energy. In the free DNA ytem, a torional energy of 233 bp can be releaed by nick. Baed on experimental and quantitative reult, we propoe a mechanim to explain the configuration tranition of thefreecirculardnaundertheinfluenceofnick.can releae the torional energy of local bp in DNA molecule. The preent finding are valuable in undertanding the behavior of nick in free DNA ytem.

2 2 Nanomaterial 2686 puc19 DNA Nb.BrDI 1765 Nt.BtNBI Two nick Four nick 1191 Six nick 1191 (c) (d) Figure 1: ing ite in puc19 DNA were produced by two nicking endonucleae. The blue and green ymbol repreent Nb.BrDI and Nt.BtNBI, repectively. Schematic of angle degree on puc19 DNA produced by two nicking endonucleae (b d). ing angle of puc19 DNA with two, (c) four, and (d) ix nick. 2. Material and Method 2.1. Material. In the experiment, puc19, a commonly ued plamid and cloning vector for Echerichia coli, wa purchaed from New England Biolab. The molecule i a double-tranded circular plamid having a length of 2,686 bp. Two nicking endonucleae, each cleaving only one trand of adouble-trandeddnaubtrate,werealopurchaedfrom New England Biolab Chemical Reagent. In the experiment, Nb.BrDI and Nt.BtNBI, two nicking endonucleae, were ued to produce nicked puc19 [13 15]. The recognition ite of two nicking endonucleae on puc19 DNA are hown in Figure 1. According to induction of New England Biolab, a total reaction volume of 5 μl containing 2 μg ofcloedcircular puc19dnaandtenunitofnb.brdiinnebbuffer2were incubated for 3 h at 65 C. The mixture wa incubated for 2 min at 8 C to quench the reaction, and the nicked puc19 wa purified from thi reaction mixture by a centrifugal filter (Millipore Microcon). The reaction buffer (2 μl) containing tenunitofnt.btnbiandcloedcircularpuc19dna(2μg) wa incubated at 55 Cfor3h.Thereactionwathenheated to 8 Cfor2minandpurifieduingtheamemethoda

3 Nanomaterial 3 decribed above. ed puc19 DNA wa alo digeted by Nt.BtNBI according to the method decribed above. ed circular puc19 DNA with a varying number of nick wa obtained with the aid of two nicking endonucleae Preparation of Sample. AFM experiment were carried out to tudy the configuration tranition of plamid DNA. All ample were performed in a olution of 1 mm Tri- HCl, ph 7.5, and the concentration of DNA ued wa.12 ng/μl[16]. For AFM imaging, a reaction volume of 2 μl containing 5 mm MgCl 2 wa ued. The mixture wa depoited on the urface of frehly cleaved mica. After 15 min, the mica urface wa wahed everal time with 2 μl of Milli-Q filtered ultrapure water and then blown dry by a gentle tream of nitrogen ga [17] AFM. In thi tudy, all image were obtained under ambient condition uing a multimode AFM with a Nanocope IIIa controller (Digital Intrument, Santa Barbara, CA, USA) in tapping mode. A ilicon RTESP14 probe from Veeco (USA) wa ued at a reonance frequency range of 314 khz to 316 khz. The E canner wa ued in the experiment. The can frequency wa 1 Hz per line, and thecanizerangedfrom1μm to4μm. All AFM image at areolutionof pixel were not modified, except to flatten them. 3. Reult and Dicuion Uing AFM, we oberved the configuration tranition of puc19 DNA in the preence of nick. Full, three-dimenional view with equilibrium conformation of DNA in olution are difficult to obtain. However, under appropriate experimental condition, DNA can adhere to a mica urface weakly enough o that the interaction between DNA and mica modified by Mg 2+ doe not affect the equilibrium conformation of DNA in two-dimenional view, unlike the interaction between DNA and mica modified by 3- aminopropyltriethoxyilane [18]. A hown in Figure 2, puc19dnacanbeditinguihedbaedonmorphological difference. puc19 molecule are circular and rarely interect. A quantitative geometric analyi for every DNA molecule wa performed to tudy the configuration tate in detail. In the AFM image, the configuration tate of the puc19 DNA can be claified into two type (flat circular and interecting form) according to the geometric hape of a ingle circular DNA molecule. The flat circular form (Figure 2) doe not interect or twit within itelf. The interecting form i demontrated in Figure 2(c). Uing two nicking endonucleae, we tudied the configuration tranition of the puc19 DNA containing nick. Figure3 demontrate configuration tranition of puc19 DNA induced by a varying number of nick. In Figure 3, there are a lot of circular DNA molecule with interection form. Then, the quantity of interection form decreae a hown in Figure 3, whichrevealtheconfiguration tranition from interection form to flat circular form under the influence of two nick. When four nick exit in the Flat circular form Interecting form 1 nm 1 nm (c) 5 nm 1 nm 1 nm Figure 2: AFM image of natural puc19 DNA in the abence of nick. Repreentative image of natural circular DNA with two configuration form. Image of the flat circular form and (c) interecting form. circular DNA, there are many DNA configuration with flat circular form and little interection form a indicated by Figure 3(c). Thi reult indicate that the occurrence of interection form i obviouly decreaed a the increae of nick number. In comparion with ix nick hown in Figure 3(d), it i not obviouly different from Figure 3(c) on the configuration form. Further, circular DNA molecule were meaured in the cloed form or with two, four, or ix nick to analyze quantitatively the configuration of every circular DNA molecule on the mica urface. A decribed above, the free circular DNA ytem ha two configuration 3 3 3

4 4 Nanomaterial nm 5 nm nm 5 nm (c). (d). Figure 3: Repreentative image of circular DNA with different number of nick. Without nick; with two nick; (c) with four nick; (d) with ix nick. tate (Figure4 and Table SI in Supplementary Material available online at A hown in Figure 4, 75% and 25% of the cloed DNA take the flat circular and interecting form, repectively. In circular DNA with two nick, the occurrence of the flat circular and interecting form i increaed to 82% and i decreaed to 18%, repectively. For puc19 DNA with four or ix nick, approximately 9% and 9% of the DNA take the flat circular and interecting form, repectively. The nicking poition recognized by the two nicking endonucleae are hown in Figure 1, wherein the nicking angle how the degree between the nicking ite and initial ite of circular DNA on topological map. The nicking angle of the puc19 DNA are illutrated in Figure 1 1(d) baed on the topological map. The nicking angle of the two nick in Figure 1 are 237 and 26,andthenickingangleof the four nick in Figure 1(c) are 59,95,16,and227. The nicking angle of ix nick are produced by two nicking endonucleae (Figure 1(d)). The angle between two nick i 23, which i le than 7% of all circular DNA molecule. Thu, the interecting tate of circular DNA occur in more than 18% of the molecule, and two nick cannot completely releae the torional energy of a DNA molecule. The angle among four nick are 168, which include three angle of 36, 65,and67.Comparedwiththeangleoffournick,theangle amongixnickionlyincreaedto33, which i le than 1% of all circular DNA molecule. Thu, the puc19 DNA with four and ix nick exhibit imilar degree of configuration tate. Moreover, the nicking angle mainly occur between 59 and 26. Thu, other angle of 16 are not affected by nick. The nicking aymmetry lead to the torional energy of circular DNA not being completely releaed. In addition, the occurrence of the interecting tate i approximately 9% in the preence of ix nick.

5 Nanomaterial 5 1 Two configuration form (%) Flat circular form Interecting form Figure 4: Quantitative reult of puc19 DNA with different number of nick. Percentage of occurrence of the two form of nicked puc19 DNA without nick (), with two nick (2), with four nick (4), and with ix nick (6) d puc19 DNA with two nick puc19 DNA with four nick d Figure 5: Schematic of the releae length on puc19 DNA produced by nick. Pink egment indicate that torional energy i completely releaed. Segment indicated by repreent the releae length of bae pair induced by nick. Segment indicated by d repreentthe ditant length between two neighbor nick, which hould be le than the double releae length by nick. The ret of puc19 DNA repreented by the black egment i the effective length. Diagram of puc19 DNA with two and four nick. According to the topological analyi of nicking angle and quantitative reult of configuration tate, we believe that local egment of circular DNA till tore torional energy. We define effective length a the torage capacity of bp for torional energy and releae length a the energy releae of bp by a nick. In the two nick hown in Figure 5, thefull length of the puc19 DNA i the um of the double releae length ( ), the length between two nick ( d ), and the effective length (black egment of the circular DNA). In the four nick hown in Figure 5, the full length of the puc19 DNA i the um of the ixfold releae length ( ), length between 441 and 77bp ( d ), and effective length (black egment of the puc19 DNA). In the definition, the effective length repreent the torage capacity of torional energy in the bae pair. The torional energy of circular DNA molecule lead to the formation of an interecting hape. We aumed that the amount of interecting hape can reflect the value of effective length. According to the quantitative reult of configuration, the occurrence of the interecting tate with two nick (18%) i approximately twice that with fournick(1%).thu,wecanaumethattheeffectivelength of two nick i alo twice that of four nick. Finally, we can calculate that the releae length i 233 bp, uggeting that the torional energy of 233 bp i completely releaed by a nick and alo demontrate the bae pair in tranmiion length of torional energy in the free DNA ytem (the calculation i decribed in the Supplementary Material). In order to tudy the phyical property of nicked DNA molecule, the effect of tenion on clutered nick in DNA molecule i tudied by applying tenion on DNA molecule with optical tweezer [19]. However, thi reearch cannot dicu the configuration tranition and torional character under the

6 6 Nanomaterial 4. Concluion (c) (d) Figure 6: Schematic of the configuration tranition of the free circular DNA ytem under the influence of nick. Torional energy of natural DNA with negative upercoil cannot be releaed and lead to configuration of interecting tate; torional energy of circular DNA can be partially releaed by a nick and the interecting form of ome DNA egment change to the flat circular form; (c) torional energy of free DNA ytem i progreively releaed by nick; (d) torional energy of free DNA ytem i completely releaed by nick, and the interecting form change to the flat circular form. influence of nick. Here, we provide the method to meaure configuration by mean of AFM obervation. Further, baed on the quantitative reult of configuration tranition, the tranmiion length of torional energy in the free DNA ytem i tudied in detail. The configuration tranition of circular DNA induced by nick can be explained a follow. In the abence of nick, natural circular DNA exhibit negative upercoiling [2] (Figure 6). In our experiment, thi torional energy account for the occurrence of interecting tate in 25% of the total configuration. According to the nicking angle of puc19 DNA topological map, two nick are near the poition of a puc19 DNA molecule that can be viewed aonenick.baedonthecalculatedreultofeffective length, one nick only partially releae a torional energy of 233 bp in the free circular DNA ytem, thereby reulting in the implification of complicated configuration with the interecting tate (Figure 6). However, local egment of circular DNA molecule till exhibit the interecting form becaue of the tranmiion limitation of torional energy. A the number of nick increae, a greater amount of torional energy in circular DNA i alo releaed, leading to further implification of the complicated configuration with interecting tate (Figure 6(c)). Finally, the number of nick i large enough to releae the torional energy of natural DNA completely (Figure 6(d)). In ummary, we developed a new method to tudy quantitatively the configuration tranition of free circular DNA ytem with different number of nick uing AFM. Free circular DNA ha two configuration tate. Configuration tranition between interecting and flat circular tate occur in the preence of different number of nick. We defined thenickingangleofdnatopologicalmapandtheeffective length of torional energy and found that a torional energy of 233 bp can be completely releaed by a nick. Furthermore, baed on experimental and quantitative reult, we propoe a mechanim that explain the configuration tranition of the free circular DNA ytem induced by nick. In the free DNA ytem, nick can releae torional energy o that the complicated configuration of interecting tate become implified. Thee reult and the preented method are important for elucidating the phyical mechanim of nick in the free DNA ytem. Conflict of Interet The author declare that there i no conflict of interet regarding the publication of thi paper. Acknowledgment Thi reearch wa upported by the National Natural Science Foundation of China (Grant ) and the Natural Science Foundation of Shandong Province of China (Grant ZR214AQ25). Reference [1] T. Lindahl, Intability and decay of the primary tructure of DNA, Nature,vol.362,no.6422,pp ,1993. [2] S. Shuman and J. Turner, Site-pecific interaction of vaccinia viru topoiomerae I with bae and ugar moietie in duplex DNA, The Biological Chemitry, vol.268,no.25,pp , [3] S. N. Huang, S. Ghoh, and Y. Pommier, Topoiomerae I alone i ufficient to produce hort DNA deletion and can alo revere nick at ribonucleotide ite, Biological Chemitry,vol.29,no.22,pp ,215. [4] E. Seeberg, L. Eide, and M. Bjørå, The bae exciion repair pathway, Trend in Biochemical Science,vol.2,no.1,pp , [5] H. Wang and J. B. Hay, Simple and rapid preparation of gapped plamid DNA for incorporation of oligomer containing pecific DNA leion, Molecular Biotechnology, vol. 19, no. 2, pp , 21. [6]L.DaviandN.Maizel, Homology-directedrepairofDNA nick via pathway ditinct from canonical double-trand break repair, Proceeding of the National Academy of Science of the United State of America, vol. 111, no. 1, pp. E924 E932, 214. [7] H. Wang and J. B. Hay, Mimatch repair in human nuclear extract. Quantitative analye of exciion of nicked circular mimatched DNA ubtrate, contructed by a new technique employing ynthetic oligonucleotide, Biological Chemitry, vol. 277, no. 29, pp , 22.

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