Expression, purification, interactions of proteins and helicase assays GST-tagged or His-tagged proteins were expressed in bacterial cells according to standard protocols at 18oC and subsequently purified by binding to either Glutathione S-Sepharose (GE

hTERT-immortalized BS fibroblasts GM03509 (referred as BS) and chromosome 15 minichromosome-corrected BS fibroblasts (referred as A-15) (donated by Jerry Shay), GFP-BLM expressing BS fibroblasts GM08505 (referred as GFP-BLM) (donated by Nathan Ellis) were maintained with DMEM supplemented with 10% FCS, glutamine, sodium pyruvate and antibiotics. hTERT-immortalised RIDDLE patient cell line expressing the HA-tagged vector (referred as Vector) or HA tagged-RNF168 corrected Vector cell lines (referred as HARNF168) were maintained as described (Stewart et al, 2009). U2OS shRNF8 and U2OS shRNF168 (donated by Jiri Lukas) were maintained as described (Doil et al, 2009; Mailand et al, 2007). Treatment with hydroxyurea (HU) was for 16 hours. Cells were exposed to IR irradiation (5Gy) and then allowed to recover for 4 hrs. LLnL (10mM, Sigma) or MG132 (10mM, Calbiochem) treatments were carried out for the last 6 hr of the HU-treatment. Parallel plates were washed and allowed to grow for a further 6 hours (post-wash condition, +HU/PW). Dox regulated shutdown of RNF8 and RNF168 was done for 48 hours before the initiation of the experiments.