Figure 4.

A 5′ phosphate (5′P) group is required for RPA-dependent inhibition of PCNA-AF488₂ closing on 5′DNA, but to not for SSB-dependent inhibition of β-AF488₂ closing on 5′DNA. Reactions were performed as outlined in Figure 1B. DNA structures are illustrated in Figure 1C, with the exception of the 5′P group, which is not present here. (A) Representative β-AF488₂ closing traces are shown for 3′DNA (dark blue), 5′DNA (green), 3′DNA·SSB (light blue) and 5′DNA·SSB (gray). Calculated observed rates of β-AF488₂ closing are reported in Table 1. (B) β closing is shown as a function of 5′DNA·SSB concentration. The DNA and SSB concentrations used were: 40 nM DNA and 320 nM SSB (green), 300 nM DNA and 3 μM SSB (dark blue). (C) Representative PCNA-AF488₂ closing time courses are shown for 3′DNA (dark blue), 5′DNA (green), 3′DNA·RPA (light blue) and 5′DNA·RPA (gray). Calculated observed rates of PCNA-AF488₂ closing are reported in Table 2.