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Published online 5 March 2001. doi:10.1083/jcb.152.5.985
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© The Rockefeller University Press, 0021-9525/2001//985 $5.00
The Journal of Cell Biology, Volume 152, Number 5, , 2001 985-996

Original Article

Evidence for a Replication Function of Ffa-1, the Xenopus Orthologue of Werner Syndrome Protein



Chin-Yi Chena, Jeanine Grahama, and Hong Yana

a Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111
Fox Chase Cancer Center, 7701 Burholme Ave., Philadelphia, PA 19111.(215) 728-3616(215) 728-2514

hong_yan{at}fccc.edu

DNA replication in higher eukaryotic cells occurs at a large number of discrete sites called replication foci. We have previously purified a protein, focus-forming activity 1 (FFA-1), which is involved in the assembly of putative prereplication foci in Xenopus egg extracts. FFA-1 is the orthologue of the Werner syndrome gene product (WRN), a member of the RecQ helicase family. In this paper we show that FFA-1 colocalizes with sites of DNA synthesis and the single-stranded DNA binding protein, replication protein A (RPA), in nuclei reconstituted in the egg extract. In addition, we show that two glutathione S-transferase FFA-1 fusion proteins can inhibit DNA replication in a dominant negative manner. The dominant negative effect correlates with the incorporation of the fusion proteins into replication foci to form "hybrid foci," which are unable to engage in DNA replication. At the biochemical level, RPA can interact with FFA-1 and specifically stimulates its DNA helicase activity. However, in the presence of the dominant negative mutant proteins, the stimulation is prevented. These results provide the first direct biochemical evidence of an important role for FFA-1 in DNA replication.

Key Words: FFA-1 • Werner syndrome • Rec Q helicase • DNA replication • Xenopus laevis

© 2001 The Rockefeller University Press

Abbreviations used in this paper: ELB, egg lysis buffer; FFA-1, focus-forming activity 1; GST, glutathione S-transferase; RPA, replication protein A; WRN, WS gene product; WS, Werner syndrome.


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