Figure 5.

HIRAN is not required for regression of forks containing ssDNA at the junction. A, schematic of fork regression assay. A model fork substrate containing a two-nucleotide mismatch at the junction to prevent spontaneous branch migration is incubated with HLTF and ATP to yield annealed template and nascent duplexes. ³²P labels are indicated by asterisks. B, representative native gel of time-dependent fork regression by HLTF and ΔHIRAN against a fork substrate with fully paired nascent arms. Endpoints of reactions containing ATPase-dead HLTF^DEAA or ΔHIRAN^DEAA proteins, or of a mock reaction with no protein are shown as negative controls. The plot shows quantitation of three experiments (mean ± S.D.). Data were fit by a single exponential function. C and D, quantitation of three time-dependent fork regression reactions (mean ± S.D.) with HLTF and ΔHIRAN using forks in which the leading or lagging nascent strands have been shortened (C) or removed (D). Mock reactions contain no enzyme. Representative gels for panels C and D are shown in Fig. S3.