FRET analysis of p66 and PCNA co-localisation FRET analysis was carried out as described in the Methods section. Cells were co-transfected with two constructs as shown. PCNA was tagged with both ECFP and EYFP shown in green and red, respectively. p66 and p66Δ N145 were tagged with EYFP (shown in red) and UNG2 with ECFP (shown in green). Five of the representative high FRET values found within the given levels of intensities (donor intensities (I1, ID1) between 85–190, and acceptor intensities (I3, IA3) between 70–190. NFRET represents FRET normalized against protein expression levels. FRET is calculated from the mean of the intensities within one region of interest (ROI) containing more than 25 pixels (i.e., one replication focus). Within ROI, all individual pixels had intensities below 250. More than 90% of the UNG2-p66 co-localising foci did not FRET (FRET<0, see the Methods section).