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 ACTG 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 ACTG 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 ACTG 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 ACTG 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 ACTG 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.