Mechanism Whereby Proliferating Cell Nuclear Antigen Stimulates Flap Endonuclease 1

There is much evidence to indicate that FEN-1 efficiently cleaves single-stranded DNA flaps but is unable to process double-stranded flaps or flaps adopting secondary structures. However, the absence of Fen1 in yeast results in a significant increase in trinucleotide repeat (TNR) expansion. There are then two possibilities. One is that TNRs do not always form stable secondary structures or that FEN-1 has an alternative approach to resolve the secondary structures. In the present study, we test the hypothesis that concerted action of exonuclease and gap-dependent endonuclease activities of FEN-1 play a role in the resolution of secondary structures formed by (CTG) n and (GAA) n repeats. Employing a yeast FEN-1 mutant, E176A, which is deficient in exonuclease (EXO) and gap endonuclease (GEN) activities but retains almost all of its flap endonuclease (FEN) activity, we show severe defects in the cleavage of various TNR intermediate substrates. Precise knock-in of this point mutation causes an increase in both the expansion and fragility of a (CTG) n tract in vivo. Taken together, our biochemical and genetic analyses suggest that although FEN activity is important for singlestranded flap processing, EXO and GEN activities may contribute to the resolution of structured flaps. A model is presented to explain how the concerted action of EXO and GEN activities may contribute to resolving structured flaps, thereby preventing their expansion in the genome.

Section: Discussionmentioning

confidence: 99%

Concerted Action of Exonuclease and Gap-dependent Endonuclease Activities of FEN-1 Contributes to the Resolution of Triplet Repeat Sequences (CTG) - and (GAA) -derived Secondary Structures Formed during Maturation of Okazaki Fragments

Singh

Zheng

Chavez

et al. 2007

“…Thus EXO-1 cleaves primarily at the base of the flap, yielding a nicked duplex intermediate that can be readily sealed by ligase. In contrast, FEN-1 (in the absence or presence of WRN) incises primarily 1 nt into the downstream annealed region, and to a lesser extent at the base of the flap, resulting in primarily a 1-nt gap duplex and a lower amount of nicked duplex, respectively (24,41). This difference may be important during Okazaki fragment processing of double flap substrates in which the upstream primer has a single unannealed nt, a preferred DNA substrate for FEN-1 cleavage (48).…”

Section: Discussionmentioning

confidence: 99%

“…Substrates were prepared as described previously (41). Briefly, 10 pmol of the appropriate flap oligonucleotide was 5Ј-radiolabeled with [␥-32 P]ATP and T4 polynucleotide kinase (New England Biolabs) using the manufacturer's protocol.…”

Section: Wrn-exo-1 Coimmunoprecipitationmentioning

confidence: 99%

The Exonucleolytic and Endonucleolytic Cleavage Activities of Human Exonuclease 1 Are Stimulated by an Interaction with the Carboxyl-terminal Region of the Werner Syndrome Protein

Sharma

Sommers

Driscoll

et al. 2003

Exonuclease 1 (EXO-1), a member of the RAD2 family of nucleases, has recently been proposed to function in the genetic pathways of DNA recombination, repair, and replication which are important for genome integrity. Although the role of EXO-1 is not well understood, its 5 to 3 -exonuclease and flap endonuclease activities may cleave intermediates that arise during DNA metabolism. In this study, we provide evidence that the Werner syndrome protein (WRN) physically interacts with human EXO-1 and dramatically stimulates both the exonucleolytic and endonucleolytic incision functions of EXO-1. The functional interaction between WRN and EXO-1 is mediated by a protein domain of WRN which interacts with flap endonuclease 1 (FEN-1). Thus, the genomic instability observed in WRN؊/؊ cells may be at least partially attributed to the lack of interactions between the WRN protein and human nucleases including EXO-1.Werner syndrome (WS) 1 is a hereditary premature aging disorder characterized by genome instability (1). WS cells display elevated chromosomal aberrations (2-4), replication defects (3, 5-8), abnormal recombination (9, 10), altered telomere dynamics (11), and hypersensitivity to DNA-damaging agents (12-16). The gene defective in WS, designated WRN (17), encodes a protein with DNA helicase (18,19) and exonuclease (20 -22) activities which presumably functions in DNA metabolism to preserve genome integrity. To understand the basis of WS, a number of groups have investigated WRN protein interactions (for review, see Ref. 23). The collective work indicates that WRN interacts functionally with proteins implicated in replication and DNA repair including replication protein A (RPA), p53, Ku, and polymerase ␦. These studies have enabled researchers to speculate about pathways of DNA metabolism in which WRN might participate; however, the precise functions of the WRN gene product in vivo are not well understood.Recently we reported a novel interaction of WRN protein with the human 5Ј-flap endonuclease/5Ј to 3Ј-exonuclease (FEN-1) (24), a DNA structure-specific nuclease implicated in DNA replication, recombination, and repair (25). WRN protein stimulates FEN-1 cleavage activity by a physical interaction with a COOH-terminal domain of the WRN protein (24). Another member of the RAD2 family to which FEN-1 belongs is human exonuclease 1 (EXO-1) (26). Like FEN-1, EXO-1 is a structure-specific endonuclease as well as an exonuclease (27, 28). EXO-1 has been implicated in a number of DNA metabolic pathways including mismatch correction, mitotic and meiotic recombination, and double strand break repair (29 -34). A role for EXO-1 in Okazaki fragment processing during DNA replication has been suggested based on its structure-specific endonuclease and RNase H activities that are similar to those of FEN-1 (30). Functional overlap between EXO-1 and FEN-1 (Rad27 in Saccharomyces cerevisiae) has been proposed from observations in yeast that exoI: rad27 double mutants are inviable (35) and that overexpression of yeast EXO-1 or human EXO-1 co...

“…However, it has been shown that PCNA can stimulate Fen1 activity up to 50-fold under physiological salt conditions (16,18). Kinetic analysis revealed that PCNA enhances Fen1 binding stability, thus increasing the cleavage efficiency (28). To efficiently stimulate Fen1 activity, the PCNA trimer must encircle the DNA and must be located "below" the flap (15).…”

Section: Both the Pcna-binding Motif And The C-terminal Tail Of Humanmentioning

confidence: 99%

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

Stucki

Jónsson²,

Hübscher

2001

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.