Figure 5.

ResT unwinds synthetic replication forks. 8% PAGE 1× TAE/0.1%SDS gel analysis of timecourse reactions incubated at 37°C with various assemblies of a replication fork mimic with heterologous arms. (A) Reactions with partial duplex (PD) substrates (B) with the splayed ends substrate (C) with the flap substrates and (D) with two alternate labeling assemblies of the full fork substrate are shown. The substrates are diagrammed and named above the gel panels. The red/shaded asterisk indicates a 5΄-endlabel and the red/shaded line indicates DNA strands that are radiolabeled, unlabeled strands are shown with a black line. M, denotes mock incubation at 37°C for 90 min without ResT addition; dn, indicates heat denaturation at 95°C for 6 min in SDS-load dye prior to gel loading. The SDS-load dye allows partial renaturation of the heated species, accounting for the multiple species indicated in those lanes. The schematics to the side of the gels indicate the gel migration position of the various products of substrate unwinding and heat denaturation/partial renaturation. Assignment of the gel migration position of the various species was verified on separate gels with alternative substrate labeling configuration and markers. Substrate was present at 15 nM and ResT at 37 nM.