PCNA stimulates catalysis by structure-specific nucleases using two distinct mechanisms: substrate targeting and catalytic step

R D Hutton, J A Roberts, Carlos Penedo, Malcolm F White

Research output: Contribution to journalArticlepeer-review

24 Citations (Scopus)
4 Downloads (Pure)

Abstract

The sliding clamp Proliferating Cell Nuclear Antigen (PCNA) functions as a recruiter and organizer of a wide variety of DNA modifying enzymes including nucleases, helicases, polymerases and glycosylases. The 5-flap endonuclease Fen-1 is essential for Okazaki fragment processing in eukaryotes and archaea, and is targeted to the replication fork by PCNA. Crenarchaeal XPF, a 3-flap endonuclease, is also stimulated by PCNA in vitro. Using a novel continuous fluorimetric assay, we demonstrate that PCNA activates these two nucleases by fundamentally different mechanisms. PCNA stimulates Fen-1 by increasing the enzymes binding affinity for substrates, as suggested previously. However, PCNA activates XPF by increasing the catalytic rate constant by four orders of magnitude without affecting the K-M. PCNA may function as a platform upon which XPF exerts force to distort DNA substrates, destabilizing the substrate and/or stabilizing the transition state structure. This suggests that PCNA can function directly in supporting catalysis as an essential cofactor in some circumstances, a new role for a protein that is generally assumed to perform a passive targeting and organizing function in molecular biology. This could provide a mechanism for the exquisite control of nuclease activity targeted to specific circumstances, such as replication forks or damaged DNA with pre-loaded PCNA.

Original languageEnglish
Pages (from-to)6720-6727
Number of pages8
JournalNucleic Acids Research
Volume36
Issue number21
Early online date23 Oct 2008
DOIs
Publication statusPublished - Dec 2008

Keywords

  • Heterotrimeric PCNA
  • Mismatch repair
  • Sulfolobus-solfataricus
  • DNA
  • Endonuclease
  • Proteins
  • Ubiquitin
  • Enzyme
  • Sumo

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