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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.


RFC releases PCNA onto the duplex region of P/T DNA. Each FRETtrace represents the average of at least eight shots. For comparison,all FRET traces are normalized to the same starting value (at t = 0.005 s). (A) Schematic representation of the experimentalprocedure for panel B. (B) Cy5-PCNA (110 nM homotrimer) was preincubatedwith RFC (110 nM) and ATP. This preformed RFC·Cy5-PCNA·ATPcomplex was mixed with Cy3-labeled DNA (100 nM Cy3P/BioT or Cy3P/T)and ATP in a stopped-flow instrument, and the FRET signal was followed.The loading traces for the Cy3P/T (gray) and Cy3P/BioT DNA (black)substrates are shown, and each was fit to a double-exponential equation.The calculated rate constants are reported for each. (C) Extendedtime courses (10 s) for each FRET trace from panel B. (D) Schematicrepresentation of the experimental procedure for panel E. (E) Theexperiments depicted in panel B were repeated except the Cy3-labeledDNA was preincubated with excess RPA (242 nM). The loading trace forthe Cy3P/T DNA substrate (gray) was fit to a double-exponential equation.The loading trace for the Cy3P/BioT DNA substrate (black) was fitto a single exponential and a linear phase. The kinetic values calculatedfrom the fits for each trace are indicated. (F) Extended time courses(10 s) for each FRET trace from panel E.
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fig3: RFC releases PCNA onto the duplex region of P/T DNA. Each FRETtrace represents the average of at least eight shots. For comparison,all FRET traces are normalized to the same starting value (at t = 0.005 s). (A) Schematic representation of the experimentalprocedure for panel B. (B) Cy5-PCNA (110 nM homotrimer) was preincubatedwith RFC (110 nM) and ATP. This preformed RFC·Cy5-PCNA·ATPcomplex was mixed with Cy3-labeled DNA (100 nM Cy3P/BioT or Cy3P/T)and ATP in a stopped-flow instrument, and the FRET signal was followed.The loading traces for the Cy3P/T (gray) and Cy3P/BioT DNA (black)substrates are shown, and each was fit to a double-exponential equation.The calculated rate constants are reported for each. (C) Extendedtime courses (10 s) for each FRET trace from panel B. (D) Schematicrepresentation of the experimental procedure for panel E. (E) Theexperiments depicted in panel B were repeated except the Cy3-labeledDNA was preincubated with excess RPA (242 nM). The loading trace forthe Cy3P/T DNA substrate (gray) was fit to a double-exponential equation.The loading trace for the Cy3P/BioT DNA substrate (black) was fitto a single exponential and a linear phase. The kinetic values calculatedfrom the fits for each trace are indicated. (F) Extended time courses(10 s) for each FRET trace from panel E.

Mentions: To monitorthe dynamics of PCNA on DNA prior to equilibrium (i.e., pre-steadystate), we preassembled a RFC·ATP·Cy5-PCNA complex and rapidlymixed it with ATP and a Cy3-labeled DNA substrate in a stopped-flowapparatus (Figure 3A). In the absence of RPA, a time course of the FRET traces for theCy3P/T and Cy3P/BioT DNA substrates looked almost identical. Eachdisplayed two distinct phases (Figure 3B). First, a very rapid, single-exponential increasein the FRET signal was observed (kinc,1), confirming that RPA is not required for RFC-catalyzed loadingof Cy5-PCNA onto the Cy3-labeled DNA substrates. This was followedby a rapid, single-exponential decrease in FRET (kdec). After ∼1.0–1.5 s, the FRET signalsstabilized and remained flat for up to 10 s (Figure 3C). Under the conditions of the assay, aFRET signal is not observed on either Cy3-labeled DNA substrate atequilibrium (Figure 2C). Hence, the flat regions observed in Figure 3 indicate that the reactions have reachedequilibrium at the zero FRET state. Altogether, these FRET studiesindicate that Cy5-PCNA initially loaded onto the Cy3-labeled DNA substratesrapidly dissociates back into solution in the absence of RPA and subsequentloadings are not observed. Thus, a Cy5-PCNA·Cy3-labeled DNA complex(i.e., loaded PCNA) is disfavored at equilibrium in the absence ofRPA.


Replication Protein A Prohibits Diffusion of the PCNASliding Clamp along Single-Stranded DNA
RFC releases PCNA onto the duplex region of P/T DNA. Each FRETtrace represents the average of at least eight shots. For comparison,all FRET traces are normalized to the same starting value (at t = 0.005 s). (A) Schematic representation of the experimentalprocedure for panel B. (B) Cy5-PCNA (110 nM homotrimer) was preincubatedwith RFC (110 nM) and ATP. This preformed RFC·Cy5-PCNA·ATPcomplex was mixed with Cy3-labeled DNA (100 nM Cy3P/BioT or Cy3P/T)and ATP in a stopped-flow instrument, and the FRET signal was followed.The loading traces for the Cy3P/T (gray) and Cy3P/BioT DNA (black)substrates are shown, and each was fit to a double-exponential equation.The calculated rate constants are reported for each. (C) Extendedtime courses (10 s) for each FRET trace from panel B. (D) Schematicrepresentation of the experimental procedure for panel E. (E) Theexperiments depicted in panel B were repeated except the Cy3-labeledDNA was preincubated with excess RPA (242 nM). The loading trace forthe Cy3P/T DNA substrate (gray) was fit to a double-exponential equation.The loading trace for the Cy3P/BioT DNA substrate (black) was fitto a single exponential and a linear phase. The kinetic values calculatedfrom the fits for each trace are indicated. (F) Extended time courses(10 s) for each FRET trace from panel E.
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fig3: RFC releases PCNA onto the duplex region of P/T DNA. Each FRETtrace represents the average of at least eight shots. For comparison,all FRET traces are normalized to the same starting value (at t = 0.005 s). (A) Schematic representation of the experimentalprocedure for panel B. (B) Cy5-PCNA (110 nM homotrimer) was preincubatedwith RFC (110 nM) and ATP. This preformed RFC·Cy5-PCNA·ATPcomplex was mixed with Cy3-labeled DNA (100 nM Cy3P/BioT or Cy3P/T)and ATP in a stopped-flow instrument, and the FRET signal was followed.The loading traces for the Cy3P/T (gray) and Cy3P/BioT DNA (black)substrates are shown, and each was fit to a double-exponential equation.The calculated rate constants are reported for each. (C) Extendedtime courses (10 s) for each FRET trace from panel B. (D) Schematicrepresentation of the experimental procedure for panel E. (E) Theexperiments depicted in panel B were repeated except the Cy3-labeledDNA was preincubated with excess RPA (242 nM). The loading trace forthe Cy3P/T DNA substrate (gray) was fit to a double-exponential equation.The loading trace for the Cy3P/BioT DNA substrate (black) was fitto a single exponential and a linear phase. The kinetic values calculatedfrom the fits for each trace are indicated. (F) Extended time courses(10 s) for each FRET trace from panel E.
Mentions: To monitorthe dynamics of PCNA on DNA prior to equilibrium (i.e., pre-steadystate), we preassembled a RFC·ATP·Cy5-PCNA complex and rapidlymixed it with ATP and a Cy3-labeled DNA substrate in a stopped-flowapparatus (Figure 3A). In the absence of RPA, a time course of the FRET traces for theCy3P/T and Cy3P/BioT DNA substrates looked almost identical. Eachdisplayed two distinct phases (Figure 3B). First, a very rapid, single-exponential increasein the FRET signal was observed (kinc,1), confirming that RPA is not required for RFC-catalyzed loadingof Cy5-PCNA onto the Cy3-labeled DNA substrates. This was followedby a rapid, single-exponential decrease in FRET (kdec). After ∼1.0–1.5 s, the FRET signalsstabilized and remained flat for up to 10 s (Figure 3C). Under the conditions of the assay, aFRET signal is not observed on either Cy3-labeled DNA substrate atequilibrium (Figure 2C). Hence, the flat regions observed in Figure 3 indicate that the reactions have reachedequilibrium at the zero FRET state. Altogether, these FRET studiesindicate that Cy5-PCNA initially loaded onto the Cy3-labeled DNA substratesrapidly dissociates back into solution in the absence of RPA and subsequentloadings are not observed. Thus, a Cy5-PCNA·Cy3-labeled DNA complex(i.e., loaded PCNA) is disfavored at equilibrium in the absence ofRPA.

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.