Figure 5. DNA-PKcs
fails to alter WRN helicase activity on forked duplex, Holliday junction
and G-tailed telomeric DNA substrates. DNA helicase
assays were carried out in the presence of the indicated proteins and DNA
substrates. (A) WRN (1 nM, lanes 2, 3, 5, 8, 9, and 11) and either
DNA-PKcs (5 nM, lanes 3, 4, 9, and 10) or RPA (5 nM, lanes 5, 6, 11, and
12) were incubated in standard reaction buffer prior to addition of a 34 bp
forked duplex (0.5 nM, lanes 1-6) or a 22 bp forked duplex (0.5 nM, lanes
7-12). (B) WRN (4 nM, lanes 2-5) or BLM (2.5 nM, lanes 8-11), and
DNA-PKcs (4 nM, lane 3; 8 nM, lane 4; 20 nM, lanes 5; 2.5 nM, lane 9; 5 nM,
lane 10; 12.5 nM, lane 11) were incubated with in HJ reaction buffer prior
to addition of Holliday junction (0.5 nM, lanes 1-11). Lane 6: DNA-PKcs
(20 nM) alone.
Lane 12: heat-denatured Holliday junction denoted with filled triangles. (C)
G-tailed duplex (0.5 nM, lanes 1-5 and 7) was incubated with WRN (7.5 nM,
lane 2-5) and DNA-PKcs (6.25 nM, lane 3; 12.5 nM, lane 4; 25 nM, lanes 5
and 7) in standard reaction buffer. Lane 6: heat-denatured G-tailed duplex
denoted by a filled triangle.
