Fig 1: The RAD18–SLF1 interaction promotes DNA fiber progression. (A) MDA-MB-231 cells were transduced to express ectopic RAD18 or GFP and treated with scrambled siRNA or siRNA targeting the 3' UTR of the endogenous RAD18. Lysates were subject to western blotting to confirm knockdown of endogenous RAD18 and reconstitution with ectopic RAD18 (left). MDA-MB-231 cells with ectopic RAD18 in combinations with dox-induced RNF168 and scrambled or BRCA1 targeted siRNA were incubated with CldU for 40 min followed by a 24 h co-incubation of HU and IdU. DNA fiber lengths were assessed and are presented as the IdU/CldU length ratio, black bar indicates median values (median numbers are also shown below). A minimum of 120 replication forks were measured for each condition. ***P < 0.001 (Mann–Whitney test) compared to the scrambled siRNA treated control. (B) The indicated RAD18 cDNA constructs and mutations were generated. RAD18 constructs and a GFP control were expressed in dox-inducible RNF168 MDA-MB-231 cells and assessed for expression by western blotting. (C) Cell lines from (B) were treated with siRNA targeting BRCA1 and RAD18 3' UTR and fibers assessed as described in (A). A minimum of 150 replication forks were measured for each cell line. ***P < 0.001 (Mann–Whitney test) compared to the GFP expressing control. (D) Dox-inducible RNF168 expressing MDA-MB-231 cells were treated with the indicated siRNAs and fibers assessed as described in (A). A minimum of 200 replication forks were measured for each condition. ***P < 0.001 (Mann–Whitney test). (E) Dox-inducible GFP or RNF168 expressing MDA-MB-231 cells were treated with 4 mM HU or vehicle for 24 h and subjected to western blotting for the indicated proteins. (F) One-ended DSBs arising at HU-induced stalled replication forks activate RNF168, which subsequently localizes 53BP1 and RAD18 to ub-H2AX. In the presence of BRCA1, HR repair ensues. In the absence of BRCA1, 53BP1 inhibits DNA end resection while RAD18 promotes BIR.