Lagging Strand DNA Synthesis at the Eukaryotic Replication Fork Involves Binding and Stimulation of FEN-1 by Proliferating Cell Nuclear Antigen

Xiangyang Li,Jun Li,John Harrington,Michael R Lieber,Peter M.J Burgers

doi:10.1074/jbc.270.38.22109

Xiangyang Li, Jun Li + Show 3 more

Open Access

https://doi.org/10.1074/jbc.270.38.22109

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Abstract

The 5'-->3'-exonuclease domain of Escherichia coli DNA polymerase I is required for the completion of lagging strand DNA synthesis, and yet this domain is not present in any of the eukaryotic DNA polymerases. Recently, the gene encoding the functional and evolutionary equivalent of this 5'-->3'-exonuclease domain has been identified. It is called FEN-1 in mouse and human cells and RTH1 in Saccharomyces cerevisiae. This 42-kDa enzyme is required for Okazaki fragment processing. Here we report that FEN-1 physically interacts with proliferating cell nuclear antigen (PCNA), the processivity factor for DNA polymerases delta and epsilon. Through protein-protein interactions, PCNA focuses FEN-1 on branched DNA substrates (flap structures) and on nicked DNA substrates, thereby stimulating its activity 10-50-fold but only if PCNA can functionally assemble as a toroidal trimer around the DNA. This interaction is important in the physical orchestration of lagging strand synthesis and may have implications for how PCNA stimulates other members of the FEN-1 nuclease family in a broad range of DNA metabolic transactions.

Highlights

In eukaryotic cells, a family of structure-specific endonucleases can be defined based on conserved domains within FEN-1, a 42-kDa enzyme that is both a 5Ј flap DNA endonuclease and a nick specific 5Ј-exonuclease [1]
As the two-hybrid method may detect both direct and indirect interactions, we turned to biochemical methods to investigate a possible interaction between yeast FEN-1 (yFEN-1) and proliferating cell nuclear antigen (PCNA)
The existence of a specific protein-protein interaction between yFEN-1 and PCNA was confirmed by affinity chromatography on PCNA beads

Summary

Introduction

A family of structure-specific endonucleases can be defined based on conserved domains within FEN-1 (flap endonuclease), a 42-kDa enzyme that is both a 5Ј flap DNA endonuclease and a nick specific 5Ј-exonuclease [1]. 1 The abbreviations used are: yFEN-1, yeast FEN-1; PCNA, proliferating cell nuclear antigen; RF, replication factor; BSA, bovine serum albumin. Consistent with its corresponding functional activity, mammalian FEN-1 shows sequence homology with the 5Ј 3 3Ј-exonuclease domain present in Escherichia coli DNA polymerase I [10].

Results

Conclusion