Research Paper Volume 2, Issue 5 pp 274—284

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.