Figure 7.

Sld2T84D stimulates annealing of ssDNA to its complementary strand. (A) A schematic for annealing. Radiolabeled ssARS1-1 (1 nM) is incubated with 2 nM cold ARS1-20C-830-869-20C top strand in the presence of Sld2T84D. The annealing of these ssDNAs results in fork-shaped dsDNA. The free radiolabeled ssARS1-1 migrates faster through a native gel than the duplex containing radiolabeled ssARS1-1. (B) Sld2T84D or Sld2 (500 nM) was mixed on ice with the substrate described in (A). The mixture was then incubated at 30°C for different amounts of time, as described in the figure. Following the incubation at 30°C, the samples were analyzed as described in ‘Materials and Methods’ section. The results from (B) were quantified and plotted as the percent dsDNA versus time in minutes (C). Annealing of ssDNA in the presence of Sld2T84D or Sld2 is significantly faster than the no protein control. (D) Sld2T84D or Sld2 (50 nM) was mixed with the substrate described in (A). The results from experiments were quantified and plotted as percent dsDNA versus protein concentration. A higher fraction of DNA is annealed with Sld2T84D compared to Sld2. (E) Sld2T84D annealing ssDNA was tested with substrates containing a 5′ or 3′ overhang. The results from experiments were quantified and plotted as percent dsDNA versus time in minutes. Sld2T84D can anneal substrates with either 5′ or 3′ overhangs. (F) Dpb11, Sld2T84D or both Dpb11 and Sld2T84D (50 nM) were mixed with the substrate described in (A) for varying amounts of time, as indicated in the figure. The results from experiments similar to (F) were quantified and plotted as the percent dsDNA versus time in minutes (G). Dpb11 displays less annealing activity than Sld2T84D. Mixing Dpb11 with Sld2T84D results in an annealing activity that is greater than either protein alone.