However, at pH values higher than
pH 12.5, DNA degradation was also observed. When the DNA–Imu3 complex was heated to 100°C for 5 min in the presence of different NaCl concentrations, separation of Imu3 from DNA was observed at 0.5 M NaCl or higher click here (Additional file 3: Figure S3). Incubation of Imu3-DNA complexes with proteinase K resulted in unbound DNA due to degradation of Imu3. To determine whether DNA exposed to Imu3 could subsequently be used for molecular biological manipulations, linear plasmid pBR322 DNA that had been previously complexed with Imu3 was purified with the QIAgen commercial kit. This DNA could be re-ligated, transformed into E. coli, and again subjected to restriction enzyme activity. The integrity
of precipitated and religated plasmid DNA was confirmed on the basis of expression of the ampicillin resistance gene among 500 analysed transformants INCB024360 in vivo (described in Methods). All procedures were also performed with DNA that had not been previously complexed as a control, and no apparent losses in quantity or quality of DNA were observed (with exception of losses originating from the DNA purification procedure) (Figure 7). Further, we found that Imu3 precipitated DNA from highly (1.5 × 10-4 fold) diluted solutions, where 1 μL (100 ng) of linear plasmid DNA was diluted in 15 mL. This procedure yielded less DNA as the control but could without doubt be optimised with appropriate protocol modification (Additional file 4). The colicin DNases and their cognate immunity proteins are known to form high affinity complexes with the DNase domain [11, 12]. In the present study, despite its two preserved histidines, as nuclease inactivation motifs that are present throughout the DNase immunity protein family, Imu3 showed no coupling with the USP protein, and Imu3 alone was shown to be sufficient for protection
of Usp-producing cells. Not unexpectedly due to the sequence similarity of Imu3 with the colicin E7 immunity protein, which was shown by Dennis et al.  to be monomeric, we demonstrated, on the basis of different experiments that Imu3 does not undergo dimerisation or multimerisation. Figure 7 Representative electromobility shift assays of re-ligated DNA previously complexed with Imu3 Verteporfin in vitro (0.8% agarose gels). Lane 1, 100 ng pUC19/EcoRI DNA; lane 2: 100 ng pUC19/EcoRI DNA purified with the QIAprep kit; lane 3: 100 ng pUC19/EcoRI DNA–Imu3 complex purified with the QIAprep kit; lane 4: ligation reaction of purified DNA; lane 5: ligation reaction of purified DNA–Imu3 complex; lane 6: restriction (EcoRI) of ligation reaction of purified DNA (from lane 4); lane 7: restriction (EcoRI) of ligation reaction of purified DNA–Imu3 complex (from lane 5). M: λ/PstI marker. To the best of our knowledge, no known functions have been described yet for the protein products of orfU1, orfU2 and orfU3 (here referred to as Imu1, Imu2 and Imu3).