Evidence for a replication function of FFA-1, the Xenopus orthologue of Werner syndrome protein.
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.
Chen
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Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
Graham
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J
Yan
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D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D002457:Cell Extracts; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D005090:Exodeoxyribonucleases; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D005799:Genes, Dominant; D008297:Male; D009154:Mutation / Q000235:genetics; D009865:Oocytes; D011233:Precipitin Tests; D011485:Protein Binding; D053484:RecQ Helicases; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D051716:Replication Protein A; D013094:Spermatozoa; D014764:Viral Proteins; D014898:Werner Syndrome / Q000378:metabolism*; D000071657:Werner Syndrome Helicase; D029867:Xenopus Proteins; D014982:Xenopus laevis / Q000378:metabolism*
D002457:Cell Extracts; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D051716:Replication Protein A; D014764:Viral Proteins; D029867:Xenopus Proteins; C060035:gp32 protein, Enterobacteria phage T4; C027729:helix-destabilizing proteins; D005090:Exodeoxyribonucleases; D000251:Adenosine Triphosphatases; C506725:RECQL protein, human; D004265:DNA Helicases; D053484:RecQ Helicases; C098785:WRN protein, human; D000071657:Werner Syndrome Helicase; C507159:WRN protein, Xenopus