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Nucleic Acids Research Advance Access originally published online on October 23, 2008
Nucleic Acids Research 2008 36(21):6720-6727; doi:10.1093/nar/gkn745
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Nucleic Acids Research, 2008, Vol. 36, No. 21 6720-6727
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Nucleic Acid Enzymes

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

Richard D. Hutton1, Jennifer A. Roberts1, J. Carlos Penedo2 and Malcolm F. White1,*

1Centre for Biomolecular Sciences and 2School of Physics, University of St Andrews, Fife KY16 9ST, UK

*To whom correspondence should be addressed. Tel: +44 1334 463432; Fax: +44 1334 462595; Email: mfw2{at}st-and.ac.uk

Received August 14, 2008. Revised October 2, 2008. Accepted October 3, 2008.

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 enzyme's 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 KM. 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.

Present address: Jennifer A. Roberts, Almac Sciences (Scotland), Elvingston Science Centre, East Lothian, UK


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