ORIGINAL PAPER Cloning, molecular characterization and expression of a DNA-ligase from a new bacteriophage: Phax1 Neda Setayesh • Saleheh Sabouri-Shahrbabak • Hamid Bakherad • Zargham Sepehrizadeh Received: 22 July 2012 / Accepted: 22 May 2013 Ó Springer Science+Business Media Dordrecht 2013 Abstract DNA ligases join 3 0 hydroxyl and 5 0 phosphate ends in double stranded DNA and are necessary for maintaining the integrity of genome. The gene encoding a new Escherichia phage (Phax1) DNA ligase was cloned and sequenced. The gene contains an open reading frame with 1,428 base pairs, encoding 475 amino acid residues. Alignment of the entire amino acid sequence showed that Phax1 DNA ligase has a high degree of sequence homol- ogy with ligases from Escherichia (vB_EcoM_CBA120), Salmonella (PhiSH19 and SFP10), Shigella (phiSboM- AG3), and Deftia (phiW-14) phages. The Phax1 DNA ligase gene was expressed under the control of the T7lac promoter on the pET-16b (?) in Escherichia coli Rossetta gami. The enzyme was then homogeneously purified by a metal affinity column. Enzymatic activity of the recombi- nant DNA ligase was assayed by an in-house PCR-based method. Keywords DNA ligase Á Phage Á Cloning Á Expression Introduction DNA ligases are one of the most important enzymes in living organisms, which play pivotal roles in some of the crucial cellular processes such as DNA replication, repair and recombination. The DNA repairing mechanism is of particular interest where nicks in double stranded DNA are repaired by catalytic activity of DNA ligase through for- mation of a phosphodiester bond between adjacent terminal 5 0 -phosphoryl and 3 0 -hydroxyl groups (Doherty and Suh 2000). DNA ligases are categorized into two groups based on the type of cofactor they use for their function: ATP- dependent DNA ligases and NAD ? -dependent DNA liga- ses. End-joining by both types of DNA ligases is catalyzed through a three-step reaction. First, a covalent bond forms between the conserved active site lysine and the adenylate group of ATP or NAD ? . This step includes formation of an enzyme-AMP intermediate and subsequent release of pyrophosphate or nicotinamide mononucleotide. The AMP nucleotide is then transferred to the 5 0 phosphate of the nicked DNA strand. Finally, 3 0 -OH group of the nicked DNA acts as a nucleophile and attacks the AMP-DNA bond to seal the phosphate backbone and release the AMP (Nishida et al. 2006). It is noteworthy that DNA ligases from viruses, bacteriophages, eukarya and archaea are included in ATP-dependent DNA ligase family (EC 6.5.1.1) while DNA ligases from bacteria and some eukaryotic viruses show similarity in their amino acid sequence with NAD ? -dependent ligase family (EC 6.5.1.2). (Timson et al. 2000; Tomkinson and Mackey 1998; Wilkinson et al. 2001). All DNA, RNA and tRNA ligases as well as the eukaryotic mRNA ‘‘capping’’ enzymes, which catalyze nucleotidyl transfers, contain a conserved active-site motif that includes a lysine residue (KxDG) (Doherty and Wigley 1999). In addition to the active-site motif, there are five other conserved motifs in these enzymes, which are termed as motifs III, IIIb, IV, V and VI. There is an acceptable degree of alignment between ATP-dependent DNA ligases isolated from different microorganisms despite the fact that the molecular mass is different for each of the isolated enzymes. The crystal structures of a number of ATP- N. Setayesh Á S. Sabouri-Shahrbabak Á H. Bakherad Á Z. Sepehrizadeh (&) Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology Research Center, Tehran University of Medical Sciences, 1417614411 Tehran, Iran e-mail: zsepehri@tums.ac.ir 123 World J Microbiol Biotechnol DOI 10.1007/s11274-013-1386-1