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Related Experiment Videos

Structure-function relationship of the eukaryotic DNA replication factor, proliferating cell nuclear antigen

K Fukuda1, H Morioka, S Imajou

  • 1Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.

The Journal of Biological Chemistry
|September 22, 1995
PubMed
Summary

Proliferating cell nuclear antigen (PCNA) acts as a molecular clamp in DNA replication. Specific domains and charged amino acids on its surface are crucial for stimulating DNA polymerase delta activity and interacting with other factors.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Proliferating cell nuclear antigen (PCNA) is vital for eukaryotic DNA replication, functioning as a processivity factor for DNA polymerase delta (pol delta).
  • PCNA's structural and functional similarity to E. coli DNA polymerase III's beta-subunit suggests a molecular clamp role in DNA synthesis.

Purpose of the Study:

  • To investigate the functional domains of human PCNA essential for stimulating replication factor C (RF-C) ATPase and pol delta-mediated DNA synthesis.
  • To elucidate the specific amino acid residues and structural features responsible for PCNA's interactions with replication factors.

Main Methods:

  • Site-directed mutagenesis was employed to alter specific regions of human PCNA.
  • Biochemical analyses were conducted to assess the impact of mutations on RF-C ATPase activity and pol delta DNA synthesis stimulation.

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Main Results:

  • Deletions at the N or C termini significantly altered PCNA's structural integrity and behavior.
  • A C-terminal stretch (Lys254-Glu256) is necessary for RF-C ATPase stimulation but not for pol delta DNA synthesis stimulation.
  • Nine internal basic amino acids are critical for pol delta DNA synthesis stimulation, consistent with a positively charged inner surface for DNA clamping.
  • Charged amino acids on exposed loops and beta-sheets at the trimeric interface are important for interactions with RF-C and pol delta, respectively.

Conclusions:

  • PCNA possesses distinct functional surfaces on its outer surface for interacting with multiple replication factors.
  • The internal surface, rich in basic amino acids, facilitates DNA clamping, while specific external loops and interfaces mediate interactions with replication factors.
  • The distinguishable side surfaces of PCNA may dictate its orientation during DNA tracking.