Figure 3.

Monitoring the retention of PCNA on DNA through FRET. (A) Schematic representation of PCNA encircling a P/T junction bound by SSB. (B) Fluorescence emission spectra in the presence of SSB. Cy3P/BioT70 DNA (100 nM), Neutravidin (400 nM), ATP (1 mM), and SSB (200 nM) were pre-equilibrated at 25°C. Cy5-PCNA (110 nM homotrimer) and RFC (110 nM) were sequentially added, the solution was excited at 514 nm, and the fluorescence emission spectra was recorded from 530 to 750 nm. The fluorescence emission intensities at 665 nm (Cy5 FRET acceptor fluorescence emission max, I₆₆₅) and 561 nm (Cy3 FRET donor fluorescence emission max, I₅₆₁) are indicated. Cy5-PCNA can be excited through FRET from Cy3P/BioT70 only when the two dyes are in close proximity (<~10 nm). This is indicated by an increase in I₆₆₅ and concomitant decrease in I₅₆₁.