Structure of the DNA Repair and Replication Endonuclease and Exonuclease FEN-1

Abstract

Flap endonuclease (FEN-1) removes 5' overhanging flaps in DNA repair and processes the 5' ends of Okazaki fragments in lagging strand DNA synthesis. The crystal structure of Pyrococcus furiosus FEN-1, active-site metal ions, and mutational information indicate interactions for the single- and double-stranded portions of the flap DNA substrate and identify an unusual DNA-binding motif. The enzyme's active-site structure suggests that DNA binding induces FEN-1 to clamp onto the cleavage junction to form the productive complex. The conserved FEN-1 C terminus binds proliferating cell nuclear antigen (PCNA) and positions FEN-1 to act primarily as an exonuclease in DNA replication, in contrast to its endonuclease activity in DNA repair. FEN-1 mutations altering PCNA binding should reduce activity during replication, likely causing DNA repeat expansions as seen in some cancers and genetic diseases.

Phosphorylation of human Fen1 by cyclin-dependent kinase modulates its role in replication fork regulation

“…As the role of Fen1 in DNA replication and repair is becoming more clear, several groups are interested to structurally characterize this enzyme, and the crystal structures of two archaeal orthologous Fen1 have so far been solved (Hosfield et al, 1998;Hwang et al, 1998). For DNA repair and replication, Fen1's structural recognition of the 5 0 flap is essential.…”

Interactions of mutant and wild-type flap endonucleases with oligonucleotide substrates suggest an alternative model of DNA binding

“…Although a similar hole is present in the Methanococcus jannaschii homologue (9), this region often appears disordered (6, 7) or as a folded loop in the structure of the Pyrococcus furiosus homologue (10). The conceptual model for flap endonuclease-DNA binding has been widely accepted (1,7,9,10,15,16). Nevertheless, this model suffers from uncertainty with regard to the orientation of the flap substrate duplex because of a lack of experimental data identifying key protein-DNA interactions.…”

“…It has been suggested that the single-stranded 5Ј tail of such substrates threads through the 5Ј nuclease (4). Several prokaryotic and archaeal flap endonuclease structures have been solved (6)(7)(8)(9)(10). Because none of the reported structures contained bound DNA substrates, the precise mode of nucleic acid binding is unclear.…”

“…In T5 5Ј nuclease, the archway delineates a hole able to accommodate a threaded single-stranded nucleic acid. Although a similar hole is present in the Methanococcus jannaschii homologue (9), this region often appears disordered (6,7) or as a folded loop in the structure of the Pyrococcus furiosus homologue (10). The conceptual model for flap endonuclease-DNA binding has been widely accepted (1,7,9,10,15,16).…”

Developing master keys to brain pathology, cancer and aging from the structural biology of proteins controlling reactive oxygen species and DNA repair

“…While WRN helicase activity is regulated by Cterminal interactions that include TRF2 (Opresko et al, 2002), Rad52 (Baynton et al, 2003) and PARP-1 (von ). An example of WRN stimulation of partner proteins includes the FEN-1 partner protein, whose structures with DNA and PCNA have been defined (Hosfield et al, 1998,Chapados et al, 2004. WRN interaction that was mapped to the winged helix domain stimulates FEN-1 nucleolytic activity, by more than 80-fold (Brosh et al, 2001b).…”