Figure 2 | Scientific Reports

Figure 2

From: DNA annealing by Redβ is insufficient for homologous recombination and the additional requirements involve intra- and inter-molecular interactions

Figure 2

C3Redβ forms rings and can anneal DNA to form the nucleoprotein filament.

(a) Electrophoretic shift mobility assay (EMSA) for Redβ-DNA complexes (wt Redβ and C3Redβ) using Cy3/Cy5 labeled complementary 50 nt ssDNA strands. All lanes have the Cy3 labeled oligo (−) except lane 2. All lanes have the Cy5 labeled complementary oligo (+) except lanes 1, 8 and 9. Lanes 8 and 9 have a non-complementary Cy5 labeled oligo (o). PA; pre-annealed (lanes 3–5) – the complementary oligos were pre-annealed before adding the indicated proteins. SA; sequentially added (lanes 6–9) – the Cy3 labeled oligo was incubated with the indicated protein before addition of the Cy5 labeled oligo. The presence of a faint band shift and slight red smear in lanes 4 and 5 indicates that the oligo preannealing did not go to completion, which Redβ completed (green band) and there was excess Cy5 oligo (red smear). The annealed nucleoprotein filament complex can be seen in lane 6 (wt Redβ) and the C3-truncation (lane 7). In lanes 8 and 9, the smear shows the weak ssDNA binding of both wt and C3-truncated Redβ to the Cy3 labeled oligo. Oligonucleotide sequences are shown in Suppl. Fig. 5. (b) Atomic Force Microscopy (AFM) images of wt Redβ, C3Redβ and N1Redβ imaged without DNA. To achieve a reasonable distribution of adherent complexes on the mica surface, input concentrations of 0.7, 2.0 and 2.6 μM respectively were used. (c) Analytical size exclusion chromatography of purified wt Redβ (black line) and C3Redβ (red line) on a Superose 6 column. The grey curve shows the molecular standards with respective sizes. Inset: Coomassie-stained SDS-PAGE gel (15%) showing purified recombinant C-terminal StrepII tagged wt Redβ and C3Redβ proteins. (d) AFM images of wt Redβ and C3Redβ. Panels I: representative images of the filaments formed by wt Redβ (upper) and C3Redβ (lower) annealing two complementary 123 mers; Panels II: histograms of the different filament species illustrated below. Panels III: histograms of filament length (nm). Panels IV: histograms of filament diameters (nm). M, mean; SD, standard deviation; n, number of particles analyzed.

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