ORF57-induced double strand breaks are the result of R-loop formation and sequestration of hTREX.

<p>(<b>A</b>) Alkaline comet assays were performed on HEK 293T cells and protein levels for each assay were assessed by western blot using a monoclonal antibody to GAPDH as a loading control; monoclonal antibody to Myc for Myc-Aly; monoclonal antibody to GFP for ORF57pMut, EGFP-RNaseH1 and EGFP-AID; polyclonal antibody to RFP for mCherry and mCherry-ORF57. (<b>B</b>) Cells were transfected with mCherry or mCherry-ORF57, or treated with 50 µM etoposide for 30 minutes as a positive control, visualised by fluorescence microscopy to confirm transfection efficiency (<b>i</b>); comet assays performed and scored using CometScore (<b>ii</b>); and tail moments calculated (<b>iii</b>). (<b>C</b>) To demonstrate R-loop formation, cells were co-transfected with mCherry-ORF57 and either pMSCVgfp::AID or pEGFP-RNH, visualised by fluorescence microscopy to confirm transfection efficiency (<b>i</b>); comet assays performed and scored using CometScore (<b>ii</b>); and tail moments calculated (<b>iii</b>). (<b>D</b>)To demonstrate sequestration of hTREX by ORF57 as the cause of R-loop formation, cells were either transfected with pEGFP-57pmut or co-transfected with mCherry-ORF57 and Myc-Aly visualised by fluorescence microscopy to confirm transfection efficiency (<b>i</b>); comet assays performed and scored using CometScore (<b>ii</b>); and tail moments calculated (<b>iii</b>). n- and <i>P</i>-values are represented for all data in the figure and error bars show the standard error from the mean.