DNA substrates
utilized in this study. The sequence of the primer
(29 nucleotides) and template (62 nucleotides) strands comprising
each DNA substrate are identical. When annealed, each DNA duplex mimics
a blocked P/T junction.42 The size of the
double-stranded P/T region (29 bp) is in agreement with the requirements
for assembly of a PCNA ring onto DNA by RFC.41,45 The single-stranded DNA (ssDNA) region (33 nucleotides) adjacent
to the 3′-end of the P/T junction is consistent with the footprint
of RPA (22–30 nucleotides).39 When
prebound to Neutravidin, the biotin attached to the 3′-end
of a template strand prevents loaded PCNA from sliding off the dsDNA,
i.e., duplex, end of the substrate. A Cy3 dye attached to the 5′-end
of a primer strand serves as a FRET donor.