(A) Intermolecular end joining assay for monitoring NHEJ in HSS. The Cy3 donor fluorophore is attached to a 100-bp DNA on the surface of a coverslip inside a microfluidic flow cell. Cy5 association with Cy3 with no FRET shows long range complex formation, followed by conversion into short range complexes, where FRET between the fluorophore pair occurs [40]. (B) Intramolecular end joining assay, where Cy3 and Cy5 fluorophores are at the ends of a DNA fragment bound to the surface with an internal biotin [40]. Cy3 and Cy5 continuously are present in a diffraction-limited spot but only show FRET when a short-range complex forms. (C) Modifications to intramolecular end joining assays for monitoring enzymatic processing of non-compatible DNA ends [42]. (i) pol λ fills gaps in substrates with resected 3′ DNA ends, detected here by incorporation of a nucleotide modified with BHQ, which quenches Cy3 fluorescence. (ii) Tdp1 removes adducts on 3′ DNA ends, in this example removing Cy3 modification at one 3′ DNA end.