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Please use this identifier to cite or link to this item: http://hdl.handle.net/10023/2103
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Title: PCNA stimulates catalysis by structure-specific nucleases using two distinct mechanisms : substrate targeting and catalytic step
Authors: Hutton, R D
Roberts, J A
Penedo, Carlos
White, Malcolm F
Keywords: Heterotrimeric PCNA
Mismatch repair
Sulfolobus-solfataricus
DNA
Endonuclease
Proteins
Ubiquitin
Enzyme
Sumo
Issue Date: Dec-2008
Citation: Hutton , R D , Roberts , J A , Penedo , C & White , M F 2008 , ' PCNA stimulates catalysis by structure-specific nucleases using two distinct mechanisms : substrate targeting and catalytic step ' Nucleic Acids Research , vol 36 , no. 21 , pp. 6720-6727 .
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.
Version: Publisher PDF
Description: This work was supported by the Biotechnology and Biological Sciences Research Council [grants BBD0014391 and BBE0146741]
Status: Peer reviewed
URI: http://hdl.handle.net/10023/2103
DOI: http://dx.doi.org/10.1093/nar/gkn745
ISSN: 0305-1048
Type: Journal article
Rights: © 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.
Appears in Collections:University of St Andrews Research
Biology Research
Biomedical Sciences Research Complex (BSRC) Research



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