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. Author manuscript; available in PMC: 2010 Mar 16.
Published in final edited form as: J Biol Chem. 2002 Oct 4;277(49):47213–47224. doi: 10.1074/jbc.M206764200

Fig. 4. Single- and double-stranded DNA cannot compete effectively with primed DNA for binding RFC.

Fig. 4

A and B, competitive DNA binding assays that measure RFC (0.016 μM) binding to a mixture of 0.02 μM 32P-labeled primer-template plus 0–1 μM unlabeled 31-mer single-stranded DNA (A, ●) or 31-mer double-stranded DNA (B, ●), 0.02 μM unlabeled primer-template plus 0–1 μM 32P-labeled ssDNA (A, ■) or dsDNA (B, ■) or 0–1 μM 32P-labeled ssDNA (A, □) or dsDNA (B, □) alone. C, RFC (0.016 μM) binding to 0.02 μM 32P-primer-template DNA plus 0–1 μM primer-template competitor (●), 0.02 μM 32P-ssDNA plus 0–1 μM ssDNA competitor (□), and 0.02 μM 32P-dsDNA plus 0–1 μM dsDNA competitor (△). The data fit to a hyperbola yield a K1/2 value of 0.051, 0.037, and 0.042 μM, respectively. D, 32P-ssDNA (0–1 μM) binding to RFC (0.016 μM) alone (■) or in the presence of 0.02 μM unlabeled primer-template (○) or dsDNA (△); K1/2 = 0.16 and 0.055 μM for ssDNA binding in the presence of primer-template and dsDNA, respectively. E, 32P-ssDNA (0.02 μM) binding to RFC (0.016 μM) in the presence of 0–0.4 μM unlabeled primer-template DNA (●) or ssDNA (□); K1/2 = 0.003 and 0.037 μM with primer-template and ssDNA competitor, respectively. F, a titration of 0.005 μM RFC with 0–0.1 μM 32P-labeled primer-template alone (●) and in the presence of 0.03 μM unlabeled primer-template (○) or ssDNA (□); K1/2 = 0.045 and 0.009 μM with primer-template and ssDNA competitor, respectively