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Replication Protein A Prohibits Diffusion of the PCNASliding Clamp along Single-Stranded DNA

View Article: PubMed Central - PubMed

ABSTRACT

Thereplicative polymerases cannot accommodate distortions to thenative DNA sequence such as modifications (lesions) to the nativetemplate bases from exposure to reactive metabolites and environmentalmutagens. Consequently, DNA synthesis on an afflicted template abruptlystops upon encountering these lesions, but the replication fork progressesonward, exposing long stretches of the damaged template before eventuallystalling. Such arrests may be overcome by translesion DNA synthesis(TLS) in which specialized TLS polymerases bind to the resident proliferatingcell nuclear antigen (PCNA) and replicate the damaged DNA. Hence,a critical aspect of TLS is maintaining PCNA at or near a blockedprimer/template (P/T) junction upon uncoupling of fork progressionfrom DNA synthesis by the replicative polymerases. The single-strandedDNA (ssDNA) binding protein, replication protein A (RPA), coats theexposed template and might prohibit diffusion of PCNA along the single-strandedDNA adjacent to a blocked P/T junction. However, this idea had yetto be directly tested. We recently developed a unique Cy3-Cy5 Forsterresonance energy transfer (FRET) pair that directly reports on theoccupancy of DNA by PCNA. In this study, we utilized this FRET pairto directly and continuously monitor the retention of human PCNA ata blocked P/T junction. Results from extensive steady state and pre-steadystate FRET assays indicate that RPA binds tightly to the ssDNA adjacentto a blocked P/T junction and restricts PCNA to the upstream duplexregion by physically blocking diffusion of PCNA along ssDNA.

No MeSH data available.


Related in: MedlinePlus

Monitoringthe retention of PCNA on DNA through FRET. (A) Schematicrepresentation of PCNA encircling a P/T junction bound by RPA. Whenloaded onto P/T DNA by RFC, the Cy5 FRET acceptor on PCNA faces theCy3 FRET donor on the P/T DNA. (B) Fluorescence emission spectra inthe presence of RPA. Cy3P/BioT DNA (100 nM), Neutravidin (400 nM),ATP (1 mM), and RPA (242 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 emissionspectrum was recorded from 530 to 750 nm. The fluorescence emissionintensities at 665 nm (Cy5 FRET acceptor fluorescence emission maximum, I665) and 561 nm (Cy3 FRET donor fluorescenceemission maximum, I561) are indicated.Cy5-PCNA can be excited through FRET from Cy3P/BioT only when thetwo dyes are in close proximity of each other (less than ∼10nm). This is indicated by an increase in I665 and a concomitant decrease in I561.(C) Characterization of the steady state FRET signal. Cy5-PCNA wasassembled onto the Cy3P/BioT DNA substrate as in panel A with variouscomponents omitted, and the FRET signal (I665/I561) was measured. As a control, RFCwas omitted (column 2). (D) Titrations of the steady state FRET signal.The Cy3P/BioT DNA substrate (100 nM with 400 nM Neutravidin) was eithersaturated with RFC (110 nM) and titrated with RPA (0–242 nM)(●) or saturated with RPA (242 nM) and titrated with RFC (30–110nM) (■). Results are plotted vs the concentration of the respectivetitrant. When RPA was titrated, the FRET signal increased linearlyand then plateaued. At the break point, the concentration of RPA (129.7nM) is roughly equivalent to the concentration of RPA binding siteswithin the assay (120 nM). When RFC was titrated, the FRET signalremained constant at a level (0.565) equivalent to that observed atsaturating concentrations of RFC and RPA (0.549).
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fig2: Monitoringthe retention of PCNA on DNA through FRET. (A) Schematicrepresentation of PCNA encircling a P/T junction bound by RPA. Whenloaded onto P/T DNA by RFC, the Cy5 FRET acceptor on PCNA faces theCy3 FRET donor on the P/T DNA. (B) Fluorescence emission spectra inthe presence of RPA. Cy3P/BioT DNA (100 nM), Neutravidin (400 nM),ATP (1 mM), and RPA (242 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 emissionspectrum was recorded from 530 to 750 nm. The fluorescence emissionintensities at 665 nm (Cy5 FRET acceptor fluorescence emission maximum, I665) and 561 nm (Cy3 FRET donor fluorescenceemission maximum, I561) are indicated.Cy5-PCNA can be excited through FRET from Cy3P/BioT only when thetwo dyes are in close proximity of each other (less than ∼10nm). This is indicated by an increase in I665 and a concomitant decrease in I561.(C) Characterization of the steady state FRET signal. Cy5-PCNA wasassembled onto the Cy3P/BioT DNA substrate as in panel A with variouscomponents omitted, and the FRET signal (I665/I561) was measured. As a control, RFCwas omitted (column 2). (D) Titrations of the steady state FRET signal.The Cy3P/BioT DNA substrate (100 nM with 400 nM Neutravidin) was eithersaturated with RFC (110 nM) and titrated with RPA (0–242 nM)(●) or saturated with RPA (242 nM) and titrated with RFC (30–110nM) (■). Results are plotted vs the concentration of the respectivetitrant. When RPA was titrated, the FRET signal increased linearlyand then plateaued. At the break point, the concentration of RPA (129.7nM) is roughly equivalent to the concentration of RPA binding siteswithin the assay (120 nM). When RFC was titrated, the FRET signalremained constant at a level (0.565) equivalent to that observed atsaturating concentrations of RFC and RPA (0.549).

Mentions: To study thedynamics of PCNA during TLS, we utilized FRET to directly monitorthe retention of PCNA at a P/T junction. A P/T DNA substrate in agreementwith the requirements for assembly of a PCNA ring onto DNA by RFCwas labeled with a 5′-Cy3 FRET donor and a 3′-biotinlabel.41,45 This substrate, herein termed Cy3P/BioT,resembles a blocked P/T junction (Figure 1). The 3′-biotin label in complexwith Neutravidin serves as a protein roadblock and prevents the loadedclamp from sliding off the duplex end of the DNA; a ssDNA region directlyabuts the P/T junction and is long enough (33 nucleotides) to accommodatea single RPA molecule. PCNA was site-specifically labeled with a Cy5FRET acceptor on the “back” face of the PCNA ring, aspreviously described.41 The Cy5 dye labelhas no effect on the interaction of PCNA with RFC.41 The Cy3P/BioT DNA substrate was tested for PCNA loadingby monitoring the steady state FRET signal in the presence of RPA(Figure 2A). Cy5-PCNAcan be excited through FRET from Cy3P/BioT only when the two dye labelsare in close proximity of each other (less than ∼10 nm). Thisis indicated by an increase in the fluorescence emission intensityat 665 nm (Cy5 FRET acceptor fluorescence emission maximum, I665) and a concomitant decrease in the fluorescenceemission intensity at 561 nm (Cy3 FRET donor fluorescence emissionmaximum, I561). A FRET signal was observedonly when both RFC and ATP were present (Figure 2B), indicating that RFC loads Cy5-PCNA ontothe Cy3P/BioT DNA substrate in an ATP-dependent manner and Cy5-PCNAis stable on the Cy3/BioT DNA substrate at equilibrium.


Replication Protein A Prohibits Diffusion of the PCNASliding Clamp along Single-Stranded DNA
Monitoringthe retention of PCNA on DNA through FRET. (A) Schematicrepresentation of PCNA encircling a P/T junction bound by RPA. Whenloaded onto P/T DNA by RFC, the Cy5 FRET acceptor on PCNA faces theCy3 FRET donor on the P/T DNA. (B) Fluorescence emission spectra inthe presence of RPA. Cy3P/BioT DNA (100 nM), Neutravidin (400 nM),ATP (1 mM), and RPA (242 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 emissionspectrum was recorded from 530 to 750 nm. The fluorescence emissionintensities at 665 nm (Cy5 FRET acceptor fluorescence emission maximum, I665) and 561 nm (Cy3 FRET donor fluorescenceemission maximum, I561) are indicated.Cy5-PCNA can be excited through FRET from Cy3P/BioT only when thetwo dyes are in close proximity of each other (less than ∼10nm). This is indicated by an increase in I665 and a concomitant decrease in I561.(C) Characterization of the steady state FRET signal. Cy5-PCNA wasassembled onto the Cy3P/BioT DNA substrate as in panel A with variouscomponents omitted, and the FRET signal (I665/I561) was measured. As a control, RFCwas omitted (column 2). (D) Titrations of the steady state FRET signal.The Cy3P/BioT DNA substrate (100 nM with 400 nM Neutravidin) was eithersaturated with RFC (110 nM) and titrated with RPA (0–242 nM)(●) or saturated with RPA (242 nM) and titrated with RFC (30–110nM) (■). Results are plotted vs the concentration of the respectivetitrant. When RPA was titrated, the FRET signal increased linearlyand then plateaued. At the break point, the concentration of RPA (129.7nM) is roughly equivalent to the concentration of RPA binding siteswithin the assay (120 nM). When RFC was titrated, the FRET signalremained constant at a level (0.565) equivalent to that observed atsaturating concentrations of RFC and RPA (0.549).
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fig2: Monitoringthe retention of PCNA on DNA through FRET. (A) Schematicrepresentation of PCNA encircling a P/T junction bound by RPA. Whenloaded onto P/T DNA by RFC, the Cy5 FRET acceptor on PCNA faces theCy3 FRET donor on the P/T DNA. (B) Fluorescence emission spectra inthe presence of RPA. Cy3P/BioT DNA (100 nM), Neutravidin (400 nM),ATP (1 mM), and RPA (242 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 emissionspectrum was recorded from 530 to 750 nm. The fluorescence emissionintensities at 665 nm (Cy5 FRET acceptor fluorescence emission maximum, I665) and 561 nm (Cy3 FRET donor fluorescenceemission maximum, I561) are indicated.Cy5-PCNA can be excited through FRET from Cy3P/BioT only when thetwo dyes are in close proximity of each other (less than ∼10nm). This is indicated by an increase in I665 and a concomitant decrease in I561.(C) Characterization of the steady state FRET signal. Cy5-PCNA wasassembled onto the Cy3P/BioT DNA substrate as in panel A with variouscomponents omitted, and the FRET signal (I665/I561) was measured. As a control, RFCwas omitted (column 2). (D) Titrations of the steady state FRET signal.The Cy3P/BioT DNA substrate (100 nM with 400 nM Neutravidin) was eithersaturated with RFC (110 nM) and titrated with RPA (0–242 nM)(●) or saturated with RPA (242 nM) and titrated with RFC (30–110nM) (■). Results are plotted vs the concentration of the respectivetitrant. When RPA was titrated, the FRET signal increased linearlyand then plateaued. At the break point, the concentration of RPA (129.7nM) is roughly equivalent to the concentration of RPA binding siteswithin the assay (120 nM). When RFC was titrated, the FRET signalremained constant at a level (0.565) equivalent to that observed atsaturating concentrations of RFC and RPA (0.549).
Mentions: To study thedynamics of PCNA during TLS, we utilized FRET to directly monitorthe retention of PCNA at a P/T junction. A P/T DNA substrate in agreementwith the requirements for assembly of a PCNA ring onto DNA by RFCwas labeled with a 5′-Cy3 FRET donor and a 3′-biotinlabel.41,45 This substrate, herein termed Cy3P/BioT,resembles a blocked P/T junction (Figure 1). The 3′-biotin label in complexwith Neutravidin serves as a protein roadblock and prevents the loadedclamp from sliding off the duplex end of the DNA; a ssDNA region directlyabuts the P/T junction and is long enough (33 nucleotides) to accommodatea single RPA molecule. PCNA was site-specifically labeled with a Cy5FRET acceptor on the “back” face of the PCNA ring, aspreviously described.41 The Cy5 dye labelhas no effect on the interaction of PCNA with RFC.41 The Cy3P/BioT DNA substrate was tested for PCNA loadingby monitoring the steady state FRET signal in the presence of RPA(Figure 2A). Cy5-PCNAcan be excited through FRET from Cy3P/BioT only when the two dye labelsare in close proximity of each other (less than ∼10 nm). Thisis indicated by an increase in the fluorescence emission intensityat 665 nm (Cy5 FRET acceptor fluorescence emission maximum, I665) and a concomitant decrease in the fluorescenceemission intensity at 561 nm (Cy3 FRET donor fluorescence emissionmaximum, I561). A FRET signal was observedonly when both RFC and ATP were present (Figure 2B), indicating that RFC loads Cy5-PCNA ontothe Cy3P/BioT DNA substrate in an ATP-dependent manner and Cy5-PCNAis stable on the Cy3/BioT DNA substrate at equilibrium.

View Article: PubMed Central - PubMed

ABSTRACT

Thereplicative polymerases cannot accommodate distortions to thenative DNA sequence such as modifications (lesions) to the nativetemplate bases from exposure to reactive metabolites and environmentalmutagens. Consequently, DNA synthesis on an afflicted template abruptlystops upon encountering these lesions, but the replication fork progressesonward, exposing long stretches of the damaged template before eventuallystalling. Such arrests may be overcome by translesion DNA synthesis(TLS) in which specialized TLS polymerases bind to the resident proliferatingcell nuclear antigen (PCNA) and replicate the damaged DNA. Hence,a critical aspect of TLS is maintaining PCNA at or near a blockedprimer/template (P/T) junction upon uncoupling of fork progressionfrom DNA synthesis by the replicative polymerases. The single-strandedDNA (ssDNA) binding protein, replication protein A (RPA), coats theexposed template and might prohibit diffusion of PCNA along the single-strandedDNA adjacent to a blocked P/T junction. However, this idea had yetto be directly tested. We recently developed a unique Cy3-Cy5 Forsterresonance energy transfer (FRET) pair that directly reports on theoccupancy of DNA by PCNA. In this study, we utilized this FRET pairto directly and continuously monitor the retention of human PCNA ata blocked P/T junction. Results from extensive steady state and pre-steadystate FRET assays indicate that RPA binds tightly to the ssDNA adjacentto a blocked P/T junction and restricts PCNA to the upstream duplexregion by physically blocking diffusion of PCNA along ssDNA.

No MeSH data available.


Related in: MedlinePlus