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. 2004 Jan 12;32(1):316–327. doi: 10.1093/nar/gkh170

Figure 2.

Figure 2

Figure 2

Strand separation activity of human YB-1 on a forked DNA duplex structure. (A) Increasing amounts of purified YB-1 proteins (indicated in ng) or 40 ng of GST were incubated with a radioactive 22 bp forked duplex under standard conditions for helicase activity (see Materials and Methods) for 30 min at 37°C. Reactions were stopped in the appropriate dye buffer and the DNA products were run on a 12% native polyacrylamide gel. The double-stranded and single-stranded structures are depicted on the left. The asterisk represents the labeled strand at its 5′ end. The triangle represents heat denatured DNA. (B) YB-1 (40 ng) was incubated with a radioactive 22 bp forked duplex as in (A), in the presence or absence of 2 mM ATP. (C) Histogram representation of the YB-1 strand separation reactions performed in (B). All experiments were done in duplicates with 40 ng of purified human YB-1. The percentage strand displacement is calculated by using the formula: c.p.m. of displaced strand/(c.p.m. of displaced strand + c.p.m. of double-stranded DNA) × 100. The c.p.m. was calculated for each band of the gel as indicated in Materials and Methods.