Thermal denaturation curves of linearised pUC19 DNA and the Imu3 protein were carried out in 5 mM cacodylic buffer (pH 6.5) using a UV-vis spectrophotometer (Cary Varian Cary 100 Bio, Australia) equipped with a thermoelectrically controlled cell holder. UV absorption was measured as a function of temperature (UV melting curves) for different ratios of linear DNA and Imu3 (0, P5091 molecular weight 0.3 and 1.0 μg per 100 ng DNA), at 260 nm. The UV melting temperature ranged from 25°C to 99°C, with a heating rate of 1°C•min-1 and an equilibration time of 1 min. The melting curves of buffer and of the Imu3 protein alone were subtracted from the melting curves of the DNA–Imu3 protein complex, providing
curves that show only the changes in the thermal stability of the DNA. Further, the influences of pH, temperature and ionic strength on the separation of the DNA–Imu3 complex were examined. The effects of pH, were examined in the range from pH 3 to pH 13. Buffers used for these pH values were the following: pH 3-5, citric buffer; pH 6, MES buffer; pH 7-9, TRIS buffer; pH 10-12, glycine/NaOH buffer; pH 13, NaOH. The impacts of Cytoskeletal Signaling inhibitor various ions on the separation of the DNA–Imu3 complex were studied as 0-1 M NaCl, 350 mM KCl, 350 mM NaSCN, 70 mM MgCl2, 0.7% buy SAR302503 SDS, 1-3 M (NH4)2SO4 and 2.3 M guanidinium thiocyanate. The effects of temperature were studied 80°C
and 95°C, with a 10 min incubation of the complex, and at 100°C, with a 5 min incubation. To examine whether Imu3 binding to DNA triggers any DNA damage, religation experiments were performed. Initially, the linear plasmid DNA (pUC19) was incubated with
the Imu3 protein Monoiodotyrosine at 37°C for 30 min, to allow for the DNA–Imu3 complex to form. The samples were subsequently purified using the QIAprep Spin Miniprep kits (QIAgen). To check DNA integrity, the linearised DNA was used for a (self) ligation reaction (Fermentas); half of the ligation mixture was transformed into E. coli DH5α, while the other half was subjected to a second restriction (EcoRI). The structural integrity of the Imu3 precipitated plasmid DNA was also investigated on the basis of detection of potential mutations within a non-selected marker, the ampicillin resistance gene. For this purpose, plasmid pBR322 carrying both tetracycline and ampicillin resistance genes was employed. Plasmid DNA was digested with PstI, with a single restriction site within the ampicillin resistance gene to yield one linear DNA fragment. Following gel electrophoresis the linear plasmid DNA was precipitated with Imu3 and centrifuged for 10 minutes at 4°C, followed by washing with 0.5 ml of TE buffer. The pellet was subsequently treated with the PCR Cleaning Kit (Thermo Scientific) and several μl of the isolate were employed for re-ligation. In control experiments, ligase was omitted.