56 Journal of Clinical and Laboratory Investigation Updates, 2014, 2, 56-62 The Cloning and Propagation of an Inverted Repeat DNA in M13 Bacteriophage in Escherichia coli

Xuefeng Pan*,1,2,3, Nan Jiang2, Liang Ding1 and Fei Duan1

1School of Basic Medicine, Hebei University, Baoding 071002, China 2School of Life Science, Beijing Institute of Technology, Beijing 100081, China 3Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China Abstract: DNA inverted repeats are abundant in all genomes with potential of adopting DNA hairpin and cruciform structures that sometimes raise genomic instabilities. This work showed that an inverted repeat DNA sequence in bovine prochymosin gene could only be cloned in M13 bacteriophage by one orientation, and propagation of this orientation in E.coli cells raised an immediate deletion of a 102-bp DNA fragment, containing partial repeat DNA and partial M13 DNA. The deletion was found to be mediated by two tandemly arranged “CTGC” sequences, one in the inverted repeat sequence and another in the M13 phage, respectively, indicating that the deletion occurred via a nascent strand slipped mis-pairing replication mechanism. We were failed in cloning the opposite orientation of the inverted repeats by screening a large number of white bacteriophage plagues, suggesting that orientation may be inviable. By contrast, the same inverted repeats can well be cloned and propagated in a pUC19 plasmid in E.coli with two orientations. These results suggested that the maintenance of the inverted repeat in vivo was tightly related to the repeat foldability subjected to its location in a DNA sequence and its replication modes used. Keywords: DNA Inverted repeat, Interspersed dinucleotide repeats, Replication, Slippage, DNA folding.

INTRODUCTION structure formation in vivo [7]. It has been well Inverted repetitive DNA sequences and AC
TG documented that non-B DNA structures, including DNA dinucleotide repeats are abundant in higher eukaryotic hairpin structures mentioned herein, form preferentially and human genomes [1-5]. Amongst which, inverted on the lagging strand template of a replication fork [11- DNA repeat sequences can potentially adopt DNA 13], while a cruciform structure formed by a double hairpin structures when it is being single stranded, or stranded inverted repeat DNA sequence in the cruciform DNA structures when the two complementary absence of DNA replication, depends mainly on the strands are extruding out of a double stranded DNA DNA context, e.g., AT rich and/or the presence or helix in vivo [6-8]. The AC
TG dinucleotide repeats absence of certain ions in the environments, which may are the most abundant dinucleotide repeats in human facilitate the melting of a DNA double helix segment. genome with capabilities of forming various non-B DNA The melting is thought to be started in the DNA conformations, including Z-DNA in vitro [4,5,9,10]. sequence corresponding to the hairpin loop, and then These non-B DNA may generate genetic instabilities in spread into repeat sequences that potentially form DNA replication, transcription, recombination and hairpin arms. This may also rely on a DNA strand repair, leading to DNA deletion, duplication etc. and/or branch migration process [7]. Pathways of maintaining cell death [11,12]. a foldable repetitive DNA sequences in vivo are the ongoing investigations [14,15]. As to the maintenance To date, the molecular pathway of forming a hairpin of an inverted repeats in vivo, two types of DNA structure by an inverted repeat DNA sequence in vivo recombination have been found to be involved [14,16], has not been fully understood. Some investigations including DNA replication slippage (illegitimate/copy suggested that the DNA context of an inverted repeat choice recombination), which is usually mediated by DNA sequence corresponding to both the hairpin loop the formation of a DNA hairpin structure by an inverted (center) and the hairpin stem, and the overall size of a repeat folding [6,17-19]. The success of a slippage hairpin structure could possibly form are critical factors DNA replication relied on the presence of two short that determine both the stabilities of a DNA hairpin tandem repeats within or around the DNA hairpin structure and the propensities of a DNA hairpin structure, which provided platforms for DNA polymerase jumping over the structure barrier, leading to a DNA deletion [9,14-18]. The second type of

recombination was homologous recombination, during *Address correspondence to this author at the School of Basic Medicine, Hebei University, Baoding 071002, China; Tel: 0086-10-68914495-805, 0086- which DNA structural-specific nucleases, such as 13582661092; E-mail: [email protected] SbcCD in E.coli [6,21] and Rad50-Mre11-Xrs2 (Nbs1)

E-ISSN: 2310-9556/14 © 2014 Pharma Publisher Cloning and Propagation of an Inverted Repeat DNA Journal of Clinical and Laboratory Investigation Updates, 2014, Vol. 2, No. 2 57 in eukaryotic cells [22,23], are required to cleave the transformed TG1 or JM101 competent cells, prepared DNA structure through making a double strand break, by using a CaCl2 method [24] and plated on the LB and then the double strand break is repaired using plates containing IPTG and X-gal for screening the homologous recombination [14,16,18]. Failures either white plaques. RF DNA (replicative DNA) from the in jumping over or in processing the DNA structural white plagues appeared on the LB-IPTG and X-gal barrier may result in cell lethality due to inability in DNA plates was extracted, and the orientation of the inverted replication. DNA insertion in M13mp19 was determined by using Pst I restriction digestion, as depicted in Figure 1B1. In this work, the cloning and propagation of an inverted DNA repeat sequence with complex features 1.3. Restriction Mapping of the Deletion Region by of AC
TG dinucleotide repeats in bovine prochymosin DNA Restriction Digestions gene were investigated in E.coli. It was found that such a DNA sequence can only be cloned in M13 by one Propagation of the M13mp19 inserts was carried orientation, while another orientation seemed to be out in LB broth for a period of time (normally 6 to 7 inviable. Propagation of the M13 derivative carrying the hours); the RF DNA was isolated by using alkaline inverted repeats in E.coli generated a 102-bp DNA extraction method as described in Molecular cloning deletion, containing partial inverted DNA repeats and [24]. DNA deletion instability was characterized by partial M13 DNA sequence. By contrast, these types of using both EcoR I and Pst I restriction digestions (as DNA deletion instability and viability problems cannot they generate same sized DNA fragment when digest be seen in a pUC19- derived plasmid in a similar the M13mp19-E-E) (Figure 1B1). The deletion was situation. Furthermore, DNA analysis supported an mapped on the M13mp19-E-E-B1 DNA by using BamH idea that the deletion instability as seen in M13 was I restriction analysis. made through a nascent strand slipped mispairing 1.4. Determination of the Deletion Junction by DNA replication mechanism. Sequencing

1. MATERIAL AND METHODS The deletion junction in mutated M13mp19-E-E-B1 1.1. Bacteria Strains, Media, DNA and Biochemicals DNA was determined by using DNA sequencing, carried out with a DNA thermal cycling sequencing kit, E.coli strains TG1 (SupEhsd
5thi
(lac-proAB) F’) purchased from Applied Biosyetems (Beijing), and an and JM101 (F’traD36lacIq
(lacZ) M15proAB) were automatic DNA sequencer. used in this work. LB broth and LB agar medium were used for cultivation of the bacterial strains, as prepared 2. RESULTS AND DISCUSSION by following the protocols described in Molecular As indicated in Figure 1A,B,C and D, a DNA Cloning [24]. Plasmid pTac5 [25] and M13mp19 [23] inverted repeat sequence in bovine prochymosin gene were DNA stocks of this laboratory; Restriction was predicted to form complex structures, including a enzymes, EcoRI, PstI, BamHI, and T4 DNA ligase are big hairpin structure composed of a 8-base loop and a the products of Promega (Beijing) and New England 53-base pair stem with a free energy of -72.35.3KCal/ Biolabs (USA) respectively. Biochemicals, IPTG, X-gal Mol by using a DNA folding program, Mfold stock solutions were the products purchased from (http://mfold.rna.albany.edu/?q=mfold/dna-folding-form) Promega (Beijing). (Figure 1C and 1D, Pan-1,). This inverted repeat 1.2. Construction of the DNA Inverted Repeats in sequence also contains two tandemly linked AC
TG M13mp19 interspersed dinucleotide repeats located in the upstream and downstream of the 8-base loop Cloning of inverted repeat sequence from a sequence “5’-acatgagc-3’”, respectively (Figure 1C and prochymosin expression plasmid pTac5 onto M13mp19 1D). The AC
TG interspersed dinucleotide repeats was carried out as the follows: pTac5 was digested are well documented as Z-DNA-adopting simple with EcoR I restriction enzyme, and an 800- bp EcoR I repetitive DNA sequence in vitro [2,4,5,9,10]. To fragment was isolated by using low-melting agarose gel understand how such a DNA sequence with complex method [24]. This EcoR I fragment was then mixed with features is maintained in DNA metabolism in vivo, and EcoR I digested M13mp19 vector, and using T4 DNA to know how these DNA features influence its cloning ligase carried out ligation. This DNA mixture was then and/or propagation in vivo, we have attempted to clone

58 Journal of Clinical and Laboratory Investigation Updates, 2014, Vol. 2, No. 2 Pan et al.

Figure 1: Organizations of the inverted DNA repeat sequence in pTac5 and M13mp19 and DNA hairpin structures adopted by the inverted repeats. A. Distribution of restriction sites in pTac5 (pUC19-derivative); B. Distribution of restriction sites in M13mp19; B1, and B2 are two orientations when the inverted repeat was inserted in the EcoRI site of M13mp19. In B1, two Pst I restriction sites were separated by the inverted repeat; and B2, two Pst I restriction sites located in the same side of the inverted repeat; C. nucleotide sequences of the inverted repeat. The region that potentially adopts a big hairpin structure with a 53-base pair stem and a 8-base loop was named as Pan-1 (D). The small hairpin composed of two interspersed dinucleotide repeat of AC and TG tracts in the hairpin stem was named as P2 (D); deletion of a 102-bp DNA fragment during the propagation of M13mp19-E-E-B1 was underlined (B). this inverted repeat DNA sequence into the EcoR I site intermediate may allow the inverted repeats to fold into of M13mp19 bacteriophage from a prochymosin certain types of non-B DNA conformations, including expressive plasmid pTac5 [25] (see the materials and hairpin structure and/or Z-DNA etc. [26-28]. As methods and Figure 1A). With considerations of that described in the materials and methods section, and in M13 replicates its DNA using a rolling cycle replication Figure 1B, two orientations in the constructs were mode, during which a long single-stranded DNA expected to be equally available by screening the white Cloning and Propagation of an Inverted Repeat DNA Journal of Clinical and Laboratory Investigation Updates, 2014, Vol. 2, No. 2 59 plaques formed by M13mp19-E-E (Figure 1B1 and significant DNA deletion instability as characterized by B2). However, in our experiments, we can only isolate using EcoR I digestion. The EcoR I fragment released one orientation by screening a large number of white from the M13 RF DNA isolates in the M13-E-E-B1 plaques in several attempts (data not shown). The white plagues was shorter in length than the EcoR I orientation we isolated was that the two Pst I sites were fragment released from pTac5 plasmid (Figure 2). As it apart cross the cloned DNA sequence, which was can be seen in Figure 2, ~100bp deletion was found to named as M13mp19-E-E-B1 (as depicted in Figure be associated with the replication of M13-E-E-B1 RF 1B1). The opposite orientation, with a name of EcoR I DNA fragment (Figure 2, lane 4 and 7). The M13mp19-E-E-B2, cannot be cloned at all. As it can be deletion was further mapped within a 220-bp BamH I seen in Figure 1B2, M13mp19-E-E-B2 constructs fragment (Figure 2 lane 3 and lane 7), in which the would introduce two extra small inverted repeats at inverted DNA repeats are located (Figure 1B1), each side of the inverted repeats (Figure 1B2); suggesting that a direct correlation of the DNA deletion generating DNA inverted repeats clusters in M13mp19- to the presence of the DNA inverted repeats. However, E-E-B2, which might interfere with the M13 by contrast, similar DNA deletion cannot be seen in propagation. pTac5 (Figure 2, lanes 2-4).

We then carried out investigations on the propagation and repeats instability of M13mp19-E-E- B1 in E.coli, and compared them with those of pTac5, a pUC19 derivative. It is known that pUC19 and M13 are replicated differently. pUC19 replicates using theta replication mode, while M13 replicates through a rolling cycle mode. Theta replication coordinates the leading and lagging strand DNA synthesis in the replication fork by forming DNA polymerase dimer [28,29]. In contrast, rolling circle DNA replication of M13 bacteriophage will have to use single DNA polymerase and replicate along a long single stranded template [23,26]. By propagating these DNA constructs in E.coli TG1 and JM101 cells, respectively, we expected to test an idea that any stable non-B DNA secondary structures formed by the inverted repeats in the replications of Figure 2: Analysis on DNA deletion by restriction digestion. M13mp19-E-E-B1 and pTac5 would be different in Lanes 1-4 are uncut pTac5 plasmid DNA, digested by Pst I terms of affecting the ongoing DNA polymerase,either it (lane 2), digested by BamH I (Lane 3), and digested by EcoR is stalled completely, resulting in cell lethal problem; or I (lane 4); M, DNA marker (pBR322/BstN I); Lane 5, 6, it is stalled transitorily, leading to DNA deletion M13mp19-E-E-B1 and M13mp19, lane 7 and 8 are instability via replication resumption [7,8] which can be M13mp19-E-E-B1 digested by EcoR I and BamH I reflected by losing either the DNA construct, or whole respectively. The BamH I fragment before the deletion in or part of the repeat DNA sequences, while the latter pTac5 and after the deletion in M13mp19 was marked with does not affect the formation of the M13 phage plague; arrows. alternatively, propagation of M13mp19-E-E-B1 DNA construct forms very small plagues or no plaques due To further understand the mechanism underlying to the vigorous repression of a non-B DNA structure on this DNA deletion instability, the RF DNA isolated from DNA replication or completely loss the capability of the M13-E-E-B1 white plaques was sequenced. DNA M13 DNA replication in vivo (Figure 1B2 and reference sequencing results showed that a 102-bp DNA [30]). Since the propagation of M13mp19-E-E-B1 fragment deletion had happened between two “CTGC” formed normal plagues either on TG1 or on JM101 sequences (Figure 1C). One of such a “CTGC” repeat lawn (data not shown), suggesting that the inverted was found to be in the inverted repeat and another was DNA repeats cloned and propagated in M13mp19 with found to be the “CTGC” sequence of a Pst I restriction a defined orientation of M13mp19-E-E-B1 in E.coli did site in the pUC19 polylinker region (Figure 1A), not cause a significant cell inviability problem. suggesting that the deletion of the 102-bp sequence However, propagation of M13-E-E-B1 showed a has been made through an inverted repeat- mediated slippage DNA replication event, which can be 60 Journal of Clinical and Laboratory Investigation Updates, 2014, Vol. 2, No. 2 Pan et al. explained with a model presented in Figure 3. As it was which will be of no effect with the displacement activity shown in Figure 3, DNA replication was stalled before of the DNA polymerase III [20,30-32]. the first AC
TG dinucleotide repeat tract, which may be due to an unusual DNA conformation, such as Z- DNA formed by the dinucleotide repeat tract in DNA replication [2,4,5,9]. In Figure 3, we suggested that the replicating nascent strand copied the “CTGC” in the inverted repeat template and then completely paused there, resulting in a stalled DNA replication with a 3’ end of “GACG” sequence (Figure 3A). Such a “GACG” end will dissociate from its DNA template and re-anneal with the “CTGC” sequence of the Pst I restriction site in the pUC19 polylinker downstream of the stalled DNA replication fork, which resumes the DNA replication (Figure 3B) [32]. In such a process, a new Pst I restriction site was reproduced by the slippage DNA replication, and resulted in a precise deletion of a 102- base DNA fragment containing part of inverted repeats and part of M13 phage (within the region of two “CTGC” repeats) (Figure 3C). Based upon this finding, we postulated that the reason why the M13-E-E-B2 was failed to be cloned may simply be explained by an unavailability of a “CTGC” short DNA repeats in Figure 3: Schematic illustration for a replication slippage carrying out a successful slippage DNA replication. mechanism. A. DNA replication was blocked by a non-B DNA However, we cannot exclude the possibility that cloning conformation (here it was suggested to be the Z-conformation of M13mp19-E-E-B2 was failed due to bring into extra adopted by the interspersed AC
TG dinucleotide repeat inverted repeats in the DNA construct, which may also tracts, hatched box), left a “GACG”3’end in the newly interfere with the DNA replication (Figure 1B2). synthesized strand, and which was used to pair with the “CTGC” sequence in the Pst I restriction site downstream the Several reports suggested that the outputs of a template strand, and the stalled DNA replication was then DNA hairpin-mediated slippage replication depends resumed successfully. B. The slppage DNA replication greatly on the folding patterns of a DNA inverted generated a deletion of a 102-bp DNA sequence containing repeats, which affect both the DNA polymerase part of inverted repeat DNA and part of M13 DNA, C. pausing and the subsequent DNA replication resumption [31,32]. Viguera and coworkers found that However, an alternative explanation for the 102-bp the DNA polymerase stalls randomly along a hairpin deletion will be to suggest that the big hairpin (Figure stem, leading to forming different 3’ends and resulting 1C and 1D, Pan-1) did not form, while the AC
TG in different links of deletion products [33]. However, in dinucleotide repeats have formed a hairpin (depicted in this case, we cannot isolate other types of deletion Figure 1C and 1D, hairpin P2). In this case, AC
TG products by screening a large number of M13mp-E-E- dinucleotide repeats adopt a hairpin stem with Z-DNA B1 white plagues, nor seen the expected deletions by conformation, such a hairpin stem stalled the DNA carrying out further rounds of propagations with the replication immediately in the “CTGC” site [4,5,9,10]. M13mp19-E-E-B1 deletion mutants in E.coli TG1 and With these considerations, we further explained the JM101 (data not shown), indicating that the DNA folding pattern of the inverted repeats folding pattern by polymerase has stalled at a very defined site rather suggesting the AC
TG dinucleotide repeats adopted randomly, therefore a slippage DNA replication using a unusual DNA conformation (Figure 3A, the DNA single type of “CTGC” in the 3’ end of the stalled polymerase stalling region is indicated by using a nascent strand could only produce a unique type of hatched box). deletion. In this regard, a feasible scenario will be that the replicative DNA polymerase was immediately The necessity of the tandemly linked short repeats stalled by the AC
TG dinucleotide repeats or by the in mediating a DNA slippage replication has been well non-B DNA conformations adopted by the repeats, documented [16,32,33]. In this case, there are more Cloning and Propagation of an Inverted Repeat DNA Journal of Clinical and Laboratory Investigation Updates, 2014, Vol. 2, No. 2 61 than 5 “5’ –CTGN-3’” sites in the DNA template. ACKNOWLEDGMENTS Amongst which, two “5’-CTGG-3’” and one “5’-CTGA- This work was once supported in part by a grant 3’” are flanked in the 3’ side of the hairpin structure, from the National High Technology Program of China one “5’-CTGT-3’” and one “5’-CTGC-3’” are embedded and a grant from The Natural Science Foundation of in the hairpin stem (Figure 1C). Theoretically these 4- Beijing, China (No. 5132014). The authors would like to base DNA sequences can all be chosen as platforms thank those people at the Institute of Microbiology, for mediating a slippage DNA replication. 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Received on 27-10-2014 Accepted on 10-11-2014 Published on 15-12-2014

DOI: http://dx.doi.org/10.14205/2310-9556.2014.02.02.4

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