4-D18, leu1-32, h2 rad60-SLD2D-SUMO, ade6-704, ura4-D18, leu1-32, h rad60-SBM1,SBM2, ade6-704, ura4-D18, leu1-32, h2 rad60-SLD2D-SUMO-M, ade6-704, ura4-D18, leu1-32, h2 This work This work This work doi:10.1371/journal.pone.0013009.t001 2 September 2010 | MK 8931 cost Volume 5 | Issue 9 | e13009 Rad60 SLDs pREP41-rad60-SLD1D was created by deleting aa 22708, pREP41-rad60-SLD2D lacked aa 33406 23303071 and pREP41-rad60SLD2D-SUMO contained the coding sequence for aa 109 of S. pombe SUMO cloned in-frame with rad60-SLD2D in 
pREP41rad60-SLD2D. rad60-SLD2D-SUMO-M was created by QuikChange site-directed mutagenesis according to the manufacturers instructions. The hus5 gene was from A Carr . with 20 mM Tris HCl pH 7.9, 150 mM NaCl, 1 mM DTT. Protein elutions were monitored with an in-line UV detector and fractions collected. Dynamic Light Scattering 50 ml samples were analyzed at 4uC using a Malvern Instruments Nano S Dynamic Light Scattering instrument. Samples were spun at 14k rpm for 10 minutes and allowed to equilibrate at collection temperature for 2 minutes prior to data collection. Scattering data were analysed for peak position and width to identify particle size and polydispersity. Analysis of DNA damage responses UV irradiation was carried out on freshly plated cells using a Stratagene Stratalinker. Ionising radiation sensitivity was assayed using a 137Cs source at a dose of 10 Gymin21. Sensitivities to hydroxyurea and methyl methanesulphonate were analysed on YE agar at the doses stated. Results Relationship of the Rad60 SLDs to ubiquitin and SUMO Rad60 has two domains at its C-terminus that were initially reported to be ubiquitin-like. However, sequence comparisons indicate that SLD2 at least, resembles SUMO more closely than ubiquitin. SLD1 has identity with S. pombe ubiquitin and SUMO of 18.4% and 19.7% respectively. For SLD2 the identity with ubiquitin and SUMO is 14.3% and 23.4% respectively. The similarity between SLD2 and SUMO is further demonstrated by the recent publication of the structure of S. pombe and human SLD2. Comparison of the structures of SUMO, ubiquitin and SLD2 and the predicted structure of SLD1 indicates similar overall structures. Interestingly, the amino acids in SLD1 and SLD2 that are the same as, or similar to, amino acids in SUMO, are, in most cases, not the same in the two domains. Microscopy Methanol-fixed cells were stained with DAPI and viewed using an Applied Precision Deltavision Spectris microscope with deconvolution software. Protein purification His-tagged proteins expressed from pET15b, were purified using Ni2+ agarose according to the manufacturer’s instructions. Equilibrium Denaturation Studies Preparation of samples: A 23995290 stock solution of guanidinium HCl was diluted to obtain a large range of denaturant concentrations using a Hamilton Microlab dispenser; 100 ml of a stock solution of SLD2 protein containing 450 mM phosphate, 9 mM DTT was added to each denaturant sample. This gave a final buffer concentration of 50 mM phosphate pH 7.0 and a protein concentration of 1 mM. The protein/denaturant solutions were pre-equilibrated at 25uC for at least three hours. Fluorescence measurements: All measurements were performed in a thermostatted cuvette holder at 25uC using Varian Cary Eclipse Fluorescence Spectrophotometer. The excitation wavelength was 280 nm, band passes were set at 5 nm for excitation and emission and the fluorescence was measured at the lmax for the denatured state of 352 nm. SLD1, but not SLD2 is required
