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. 2020 Aug 14;48(16):9161–9180. doi: 10.1093/nar/gkaa660

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Published by Oxford University Press on behalf of Nucleic Acids Research 2020.

This work is written by (a) US Government employee(s) and is in the public domain in the US.

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Figure 7. — FANCJ promotes three-stranded DNA branch-migration in the 5′ to 3′ direction. DNA products of branch-migration reaction mixtures containing a radiolabeled three-stranded DNA substrate FANCJ, and ATP as indicated in Materials and Methods were incubated for the indicated times and resolved by electrophoresis on non-denaturing 8% polyacrylamide gels and visualized by autoradiography. DNA substrates were designed to detect 5′ to 3′ branch-migration activity (Panel A) or 3′ to 5′ branch-migration activity (Panel B). Representative results from at least three independent experiments are shown. Quantitation of DNA products for Panels (A) and (B) is shown in Panel (C). Standard deviations are indicated by error bars. Panel (D), FANCJ-WT or FANCJ-K52R (ATPase-deficient mutant) was incubated with 5′ to 3′ branch-migratable substrate in presence of ATP for 30 min and products resolved by gel electrophoresis. Panel (E), Quantitation of at least three experiments from Panel (D). Panel (F), Depiction of experiments to distinguish whether helicase or branch-migration is responsible for the product observed in Panel (A). The ssDNA added to the branch-migratable substrate used in Panel (A) anneals to the upstream template strand (both entirely red). However, for the helicase assay the 5′ sequence (grey) of the ssDNA oligonucleotide was changed such that it can only anneal after the 2-stranded DNA substrate was unwound first. Panel (G), FANCJ-WT was incubated with the helicase substrate in the presence of ATP and a third partially complementary ssDNA oligonucleotide as depicted in the helicase activity schematic shown in Panel (F). Note duplex length of substrate on left is 63 bp; as a control for FANCJ catalytic activity, a 19 bp forked duplex substrate was tested for unwinding by FANCJ on the right.