Fig. 5: Interactions between the N- and C-terminal halves of Rad52 influence ssDNA binding interactions.

A Schematic of the N- and C-terminal truncations of Rad52. B Mass photometry analysis shows Rad52^ΔC as a predominant single species in solution with mass corresponding to a homodecamer (254.7 ± 11.9 kDa). The complex remains a homodecamer when bound to ssDNA. C Trp. quenching analysis of Rad52^ΔC binding to poly(dT) reveals high-affinity monophasic ssDNA binding with site-size of 55 ± 3.2 nt/decamer. D Fluorescence anisotropy analysis of Rad52^ΔC binding to a FAM-(dT)₃₅ oligonucleotide shows stoichiometric high-affinity binding to ssDNA. E Fluorescence anisotropy ssDNA binding analysis of N-terminal truncated versions of Rad52 show no ssDNA binding activity for Rad52^ΔΝ, but weak binding for Rad52^ΔN*. F When Rad52^ΔN is premixed with Rad52, a reduction in the Trp quenching signal upon ssDNA (dT)₈₄ binding is observed. The signal corresponds to the loss of ssDNA binding to the low affinity site in Rad52. G When Rad52^ΔC and Rad52^ΔN are premixed and binding to ssDNA (dT)₈₄ is assessed through Trp. quenching, an initial increase in binding is observed as the high-affinity site is occupied. This is followed by a sharp loss in binding. H When similar experiments are performed by sequential addition, titration of ssDNA (dT)₈₄ to Rad52^ΔC produces an increase in Trp quenching as expected (purple data points). After addition of ssDNA, increasing concentrations of Rad52^ΔΝ* was added, leading to loss in Trp quenching. These data show that defined regions in the C-terminal half of Rad52 modulate ssDNA binding interactions. Data are representative of at least 3 independent replicates and error bars shown are +/− SEM for each data point. Source data are provided as a Source data file.