Monitoring the Retention of Human Proliferating Cell Nuclear Antigen at Primer/Template Junctions by Proteins That Bind Single-Stranded DNA

Abstract

In humans, proliferating cell nuclear antigen (PCNA) sliding clamps encircling DNA coordinate various aspects of DNA metabolism throughout the cell cycle. A critical aspect of this is restricting PCNA to the vicinity of its DNA target site. For example, PCNA must be maintained at or near primer/template (P/T) junctions during DNA synthesis. With a diverse array of cellular factors implicated, many of which interact with PCNA, DNA, or both, it is unknown how this critical feat is achieved. Furthermore, current biochemical assays that examine the retention of PCNA near P/T junctions are inefficient, discontinuous, and qualitative and significantly deviate from physiologically relevant conditions. To overcome these challenges and limitations, we recently developed a novel and convenient Förster resonance energy transfer (FRET) assay that directly and continuously monitors the retention of human PCNA at a P/T junction. Here we describe in detail the design, methodology, interpretation, and limitations of this quantitative FRET assay using the single-stranded DNA-binding protein, SSB, from Escherichia coli as an example. This powerful tool is broadly applicable to any single-stranded DNA-binding protein and may be utilized and/or expanded upon to dissect DNA metabolic pathways that are dependent upon PCNA.

Figure 1

Schematic representation of a Cy5-labeled PCNA ring loaded onto a Cy3-labeled P/T DNA substrate by RFC. The front face (orange) of the PCNA ring faces the P/T junction. The Cy5 FRET acceptor is located on the back face (green) of the PCNA ring and faces the Cy3 FRET donor at the duplex end of the P/T DNA substrate.

Figure 2

Cy3P/BioT DNA substrate for SSB. The duplex region is identical to that described previously for RPA and the size (29 bp) is in agreement with the requirements for assembly of a PCNA ring onto DNA by RFC^,. The length (70 nt) of the 5′ polyT ssDNA overhang shown in red will accommodate only a single SSB molecule at 200 mM ionic strength and permits RFC-catalyzed loading of PCNA. When pre-bound to Neutravidin, the biotin attached to the 3′-end of the template strand prevents loaded PCNA from sliding off the duplex end. A Cy3 dye attached to the 5′end of the primer strand serves as the FRET donor.

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.

Figure 4

Titrations of the steady-state FRET. The Cy3P/BioT70 DNA substrate (100 nM with 400 nM Neutravidin), ATP (1 mM), and Cy5-PCNA were held constant (110 nM homotrimer) and either saturated with RFC (110 nM) and titrated with SSB (0 – 180 nM) ( ) or saturated with SSB (180 nM) and titrated with RFC (30 – 110 nM) (·). Results are plotted versus the concentration of the respective titrant. When SSB was titrated, the FRET signal increased linearly with SSB concentration up to 90 nM. Data points within this concentration range (0 – 90 nM SSB) were fit to a linear equation. After 90 nM, the FRET plateaus and remains constant with SSB concentration. Data points within this concentration range (120 – 180 nM) were fit to a flat line. The two lines intersect at the “breakpoint” where the Cy3P/BioT70 DNA substrate is saturated with Cy5-PCNA. When RFC was titrated, the FRET signal remained constant at a level (0.557) equivalent to that observed at saturating concentrations of RFC and SSB (0.549). These FRET values are identical to those observed for RPA (Table S1). This suggests that Cy5-PCNA encircling a P/T junction is in the same FRET state when the adjacent ssDNA is bound by either RPA or SSB.

Figure 5

Characterization of the steady state FRET signal. Cy5-PCNA was assembled onto the Cy3P/BioT70 DNA substrate with various components omitted and the FRET signal (I₆₆₅/I₅₆₁) was measured.

Figure 6

Competitive inhibition of PCNA loading. A ssDNA-binding protein may competitively inhibit RFC-catalyzed loading of PCNA by binding to PCNA (panel A) or a P/T junction (panel B) with an affinity described by K_i(inhibition constant). The IC₅₀ is the concentration of the ssDNA-binding protein that decreases PCNA loading by 50% and is related to K_i by the exact solution provided by Munson and Rodbard. (A) Competitive inhibition of PCNA loading by binding to PCNA. The IC₅₀’s were calculated based on the binding affinity (K_D) of RFC for PCNA (0.2 nM) and plotted versus K_i. The IC₅₀ for a K_i of 20 nM is indicated. (B) Competitive inhibition of PCNA loading by binding to a P/T junction. The IC₅₀’s were calculated based on the binding affinity (K_D) of an RFC·PCNA complex for a P/T junction (5 nM) and plotted versus K_i. The IC₅₀ for a K_i of 50 nM is indicated.