Fig. 4
From: Structural basis for DNA unwinding at forked dsDNA by two coordinating Pif1 helicases
Single turnover unwinding of a dual duplex substrate by BaPif1 and its variants. a The substrate contains a fluorescein labeled forked 16-bp duplex (blue) called parental duplex and a Cy5 labeled 16-bp duplex on the 3′ arm of the fork (red) termed the leading duplex. b The substrate (S), intermediates (ILD with the leading duplex unwound and IPD with the parental duplex unwound) and products (PPD is the trapped product of parental duplex unwinding and PLD is the trapped product of leading duplex unwinding) as separated by native PAGE. c Unwinding assay with wild type and mutants, H236A, F379A, H377A, and Y91A/Y412A. d Unwinding assay with wild type and interface mutants, D209A, E323A/K405A, D209A/K405A and E323K. e Quantification of the products formed from leading/parental duplex region in single turnover unwinding assays with wild type BaPif1 and its variants. Values are the average product formation and standard deviation of three independent experiments. Source gels and quantification data are provided as a Source Data file.
