A Novel Role in DNA Metabolism for the Binding of Fen1/Rad27 to PCNA and Implications for Genetic Risk

Gary, Ronald K.; Park, Min; Nolan, John P.; Cornelius, Helen L.; Kozyreva, Olga G.; Tran, Hiep T.; Lobachev, Kirill S.; Resnick, Michael A.; Gordenin, Dmitry A.

doi:10.1128/mcb.19.8.5373

Cited by 97 publications

(131 citation statements)

References 55 publications

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“…For instance, Resnick and colleagues (25,26) have used a FEN1 mutant that harbors a D to A substitution at a residue found to be critical for DNA processing to determine the in vivo substrates of the flap endonuclease. Using either genetically engineered yeast strains or exogenously expressing yeast systems, they show that the FEN1 mutant causes genetic instability and increased sensitivity to MMS, likely by preventing processing of DNA replication intermediates and promoting the formation of recombinogenic DNA double-strand breaks.…”

Section: Discussionmentioning

confidence: 99%

“…For example, such a mutation in the flap endonuclease, FEN1 (i.e., a D to A substitution), resulted in a dominant-negative form of the human and yeast protein that bound substrate DNAwith high affinity and blocked subsequent nucleic acid processing in vivo (25,26). Prior studies have established the importance of the acidic residues E96 and D210 to the enzymatic repair activity of APE1 ( Fig.…”

Section: Ap Endonuclease and Dna Binding Properties Of The Ed Proteinmentioning

confidence: 99%

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A Dominant-Negative Form of the Major Human Abasic Endonuclease Enhances Cellular Sensitivity to Laboratory and Clinical DNA-Damaging Agents

McNeill

Wilson

2007

Molecular Cancer Research

109

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Apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is the primary enzyme in mammals for the repair of abasic sites in DNA, as well as a variety of 3 ¶ damages that arise upon oxidation or as products of enzymatic processing. If left unrepaired, APE1 substrates can promote mutagenic and cytotoxic outcomes. We describe herein a dominant-negative form of APE1 that lacks detectable nuclease activity and binds substrate DNA with a 13-fold higher affinity than the wild-type protein. This mutant form of APE1, termed ED, possesses two amino acid substitutions at active site residues Glu 96 (changed to Gln) and Asp 210 (changed to Asn). In vitro biochemical assays reveal that ED impedes wild-type APE1 AP site incision function, presumably by binding AP-DNA and blocking normal lesion processing. Moreover, tetracycline-regulated (tet-on) expression of ED in Chinese hamster ovary cells enhances the cytotoxic effects of the laboratory DNA-damaging agents, methyl methanesulfonate (MMS; 5.4-fold) and hydrogen peroxide (1.5-fold). This MMS-induced, ED-dependent cell killing coincides with a hyperaccumulation of AP sites, implying that excessive DNA damage is the cause of cell death. Because an objective of the study was to identify a protein reagent that could be used in targeted gene therapy protocols, the effects of ED on cellular sensitivity to a number of chemotherapeutic compounds was tested. We show herein that ED expression sensitizes Chinese hamster ovary cells to the killing effects of the alkylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (also known as carmustine) and the chain terminating nucleoside analogue dideoxycytidine (also known as zalcitabine), but not to the radiomimetic bleomycin, the nucleoside analogue B-D-arabinofuranosylcytosine (also known as cytarabine), the topoisomerase inhibitors camptothecin and etoposide, or the cross-linking agents mitomycin C and cisplatin. Transient expression of ED in the human cancer cell line NCI-H1299 enhanced cellular sensitivity to MMS, 1,3-bis(2-chloroethyl)-1-nitrosourea, and dideoxycytidine, demonstrating the potential usefulness of this strategy in the treatment of human tumors.

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Section: Discussionmentioning

confidence: 99%

Section: Ap Endonuclease and Dna Binding Properties Of The Ed Proteinmentioning

confidence: 99%

A Dominant-Negative Form of the Major Human Abasic Endonuclease Enhances Cellular Sensitivity to Laboratory and Clinical DNA-Damaging Agents

McNeill

Wilson

2007

Molecular Cancer Research

109

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“…Since protein-protein interactions can modulate the enzyme activities, e.g. PCNA can enhance the processivity of DNA polymerase ␦ and FEN1 activity (35,72,73), experiments to study the effects of APE, PCNA, and RPA on hMYH glycosylase activity are underway. …”

Section: Discussionmentioning

confidence: 99%

Human Homolog of the MutY Repair Protein (hMYH) Physically Interacts with Proteins Involved in Long Patch DNA Base Excision Repair

Parker¹,

Gu²,

Mahoney

et al. 2001

Journal of Biological Chemistry

200

207

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The human MutY homolog (hMYH) is a DNA glycosylase involved in the removal of adenines or 2-hydroxyadenines misincorporated with template guanines or 7,8-dihydro-8-oxodeoxyguanines. hMYH is associated in vivo with apurinic/apyrimidinic endonuclease (APE1), proliferating cell nuclear antigen (PCNA), and replication protein A (RPA) in HeLa nuclear extracts as shown by immunoprecipitation and Western blotting. However, binding of hMYH to DNA polymerases ␤ and ␦ was not detected. By using constructs containing different portions of hMYH fused to glutathione S-transferase, we have demonstrated that the APE1-binding site is at a region around amino acid residue 300, that the PCNA binding activity is located at the C terminus, and that RPA binds to the N terminus of hMYH. A peptide consisting of residues 505-527 of hMYH that contains a conserved PCNA-binding motif binds PCNA, and subsequent amino acid substitution identified Phe-518 and Phe-519 as essential residues required for PCNA binding. RPA binds to a peptide that consists of residues 6 -32 of hMYH and contains a conserved RPA-binding motif. The PCNA-and RPA-binding sites of hMYH are further confirmed by peptide and antibody titration. These results suggest that hMYH repair is a long patch base excision repair pathway.

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“…The amounts of Fen1 were 0.1 ng (2.4 fmol), 1 ng (24 fmol), and 10 ng (240 fmol), respectively. C, depletion of the assay reveals that Fen1 activity is dependent on the presence of both PCNA and RF-C. data using yeast strains with mutations within this conserved region (21,30). In another report, it was suggested that the basic C-terminal tail of Fen1 was also important for the interaction with PCNA, because a deletion mutant lacking 17 amino acids at the C terminus failed to interact with PCNA, although this mutant still contained an intact PCNA interaction motif (17).…”

Section: Discussionmentioning

confidence: 99%

In Eukaryotic Flap Endonuclease 1, the C Terminus Is Essential for Substrate Binding

Stucki

Jónsson²,

Hübscher

2001

Journal of Biological Chemistry

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Flap endonuclease 1 (Fen1) is a structure-specific metallonuclease with important functions in DNA replication and DNA repair. It interacts like many other proteins involved in DNA metabolic events with proliferating cell nuclear antigen (PCNA), and its enzymatic activity is stimulated by PCNA in vitro. The PCNA interaction site is located close to the C terminus of Fen1 and is flanked by a conserved basic region of 35-38 amino acids in eukaryotic species but not in archaea. We have constructed two deletion mutants of human Fen1 that lack either the PCNA interaction motif or a part of its adjacent C-terminal region and analyzed them in a variety of assays. Remarkably, deletion of the basic Cterminal region did not affect PCNA interaction but resulted in a protein with significantly reduced enzymatic activity. Electrophoretic mobility shift analysis revealed that this mutant displayed a severe defect in substrate binding. Our results suggest that the C terminus of eukaryotic Fen1 consists of two functionally distinct regions that together might form an important regulatory domain.Fen1 (5Ј exonuclease-1 or flap endonuclease-1) is a multifunctional structure-specific metallonuclease that is important for DNA metabolic events such as replication and repair. Its main function in replication is proposed to be the removal of the displaced RNA-DNA primers synthesized by DNA polymerase ␣-primase during discontinuous lagging strand replication (reviewed in Ref.

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A Novel Role in DNA Metabolism for the Binding of Fen1/Rad27 to PCNA and Implications for Genetic Risk

Cited by 97 publications

References 55 publications

A Dominant-Negative Form of the Major Human Abasic Endonuclease Enhances Cellular Sensitivity to Laboratory and Clinical DNA-Damaging Agents

A Dominant-Negative Form of the Major Human Abasic Endonuclease Enhances Cellular Sensitivity to Laboratory and Clinical DNA-Damaging Agents

Human Homolog of the MutY Repair Protein (hMYH) Physically Interacts with Proteins Involved in Long Patch DNA Base Excision Repair

In Eukaryotic Flap Endonuclease 1, the C Terminus Is Essential for Substrate Binding

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