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.

Authors

Hedglin, Mark; Aitha, Mahesh; Benkovic, Stephen J;

Keywords

  • Avidin/ chemistry
  • Avidin/ metabolism
  • Binding Sites
  • Biotin/ chemistry
  • Biotin/ metabolism
  • Carbocyanines/ chemistry
  • DNA Primers/ chemistry
  • DNA Primers/ metabolism
  • DNA Replication
  • DNA, Single-Stranded/ chemistry
  • DNA, Single-Stranded/ metabolism
  • DNA-Binding Proteins/ chemistry
  • DNA-Binding Proteins/ genetics
  • DNA-Binding Proteins/ metabolism
  • Escherichia coli Proteins/ chemistry
  • Escherichia coli Proteins/ genetics
  • Escherichia coli Proteins/ metabolism
  • Fluorescence Resonance Energy Transfer
  • Fluorescent Dyes/ chemistry
  • Humans
  • Microscopy, Fluorescence
  • Models, Molecular
  • Molecular Conformation
  • Mutation
  • Peptide Fragments/ chemistry
  • Peptide Fragments/ genetics
  • Peptide Fragments/ metabolism
  • Proliferating Cell Nuclear Antigen/ chemistry
  • Proliferating Cell Nuclear Antigen/ genetics
  • Proliferating Cell Nuclear Antigen/ metabolism
  • Protein Interaction Domains and Motifs
  • Protein Stability
  • Recombinant Proteins/ chemistry
  • Recombinant Proteins/ metabolism
  • Replication Protein A/ chemistry
  • Replication Protein A/ genetics
  • Replication Protein A/ metabolism

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