Fig. 4
From: Rad52 prevents excessive replication fork reversal and protects from nascent strand degradation
RAD52 promotes formation of a reversal-resistant fork structure. a, b Electrophoretic mobility shift analysis of the RPA and RAD52 binding to G1 DNA (a) and a model replication fork, RF1 (b). DNA substrates were incubated with the indicated concentrations of RPA and RAD52. The complexes and the unbound DNA were then separated by electrophoresis in 0.8% TAE agarose. c, d Single-molecule analysis of the replication fork-like structures in the presence of RPA and RAD52. RF2 (c), which reports on the conformation and motion of the leading strand (black) arm relative to the lagging strand arm tethered to the surface, and RF3 (d), which reports on the relative position and dynamics of the parental duplex (black) relative to the surface-tethered lagging strand arm, were immobilised on the surface of the TIRFM flow cell. The Cy3 dye (green circle) was excited by 532 nm TIR illumination, while the Cy5 dye (red circle) was excited via Förster resonance energy transfer (FRET) from Cy3. Fluorescence of the Cy3 and Cy5 dyes was recorded separately and used to calculate FRET efficiency. FRET distributions in the left column of each panel were obtained from combining three short movies and represent the overall FRET states of each substrates under the indicated conditions. Dotted green lines show the FRET distribution for each substrate in the presence of RPA only overlaid over the distribution in the presence of RPA and RAD52. Regions of the distributions marked by a blue star indicate states where the Cy5-labelled arm is brought close to the Cy3-labelled lagging strand arm. Movies recorded for 1 min were used to evaluate the conformational dynamics of the RF2 and RF3 under each experimental condition. Representative single-molecule FRET trajectories are shown in the right column by their respective FRET distributions. Source data are provided as a Source Data file
