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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.
Figure 1. Colocalization of FFA-1 and RPA in nuclei reconstituted around sperm chromatin in the interphase egg extract. Representative nuclei from early (40â45 min; AâD), middle (45â85 min; EâH), and late (85â150 min; IâL) stages are shown. (A, E, and I) FFA-1 staining. (B, F, and J) RPA staining. (C, G, and K) Merge of FFA-1 staining and RPA staining. (D, H, and L) DNA staining by Hoechst.
Figure 2. Colocalization of FFA-1 and sites of DNA synthesis. (AâF) In normal reconstituted nuclei. Biotin-dCTP was added 5 min before fixation. (AâC) 45 min. (DâF) 60 min. (GâL) In reconstituted nuclei formed in the presence of aphidicolin. (GâI) An aphidicolin-arrested nucleus directly fixed and stained. Biotin-dCTP was added to the reaction at the beginning. (HâL) An aphidicolin-arrested nucleus pelleted through 1 M sucrose cushion and then labeled with dATP, dGTP, TTP, and biotin-dCTP. (A, D, G, and J) FFA-1 staining. (B, E, H, and K) Biotin staining. (C, F, I, and L) Merge of FFA-1 staining and biotin staining.
Figure 3. The effect of FFA-1 depletion on DNA replication and RPA focus formation. (A) Western blot for FFA-1 in the extracts depleted with either antiâFFA-1 antibody or control antibody. Arrowhead indicates the position of FFA-1. (B) DNA synthesis in nuclei reconstituted around sperm chromatin with the depleted extracts. Samples were taken at the times indicated. (C and D) Immunofluorescence staining for RPA on sperm chromatin incubated in either FFA-1âdepleted (C) or control-depleted cytosol (D).
Figure 4. Effect of GSTâFFA-1 fusion protein on DNA replication. (A) Replication of nuclei reconstituted around sperm chromatin in the presence of various fusion proteins (500 nM final concentration for GST-Xho and GST-Stu/Xho; 1 μM for GST-Stu and GST) or buffer. The fusion proteins and [32P]dATP were added at the beginning of the reactions. Samples were taken at the indicated times and separated on 0.8% agarose/TAE gel. The top band is the DNA that was too large to enter the gel and the bottom band is the sheared DNA (>20 kb) introduced during sample preparation. (B) Diagram of the GSTâFFA-1 fusion proteins used in this study. Numbers indicate the positions of the amino acids that demarcate the various domains in FFA-1. (C) The fusion proteins used in this study separated on a 12% SDS polyacrylamide gel and stained by Coomassie blue. Molecular weight markers are labeled on the right in kilodaltons.
Figure 5. Formation of hybrid foci. (AâF) Nuclei were reconstituted around sperm chromatin for 52 min in the presence of GST-Xho and stained with the affinity-purified anti-GST (A and D), RPA (B), or FFA-1 (E). (C) Merge of GST staining and RPA staining. (F) Merge of GST staining and FFA-1 staining. The antiâFFA-1 antibody is against the COOH-terminal end of FFA-1 and does not react with GST-Xho. (GâO) Nuclei reconstituted in the presence of GST-Stu/Xho (GâI), GST-Stu (JâL), and GST (MâO) for 52 min. (G, J, and M) GST staining (green). (H, K, and N) RPA staining (red). (I, L, and O) Merge of GST and RPA staining.
Figure 6. Interaction between FFA-1 and RPA. (A) Coimmunoprecipitation of FFA-1 and RPA. Western blot analysis of the proteins brought down from the cytosol by the Affi-gel protein A beads precoated with the indicated antibodies. Blots were probed with the rabbit antiâFFA-1C (top) and rabbit anti-RPA (bottom). (B) Mapping of the RPA interaction domain in FFA-1. The various GSTâFFA-1 fusion proteins were incubated with the cytosol and then brought down by glutathione beads. The bound proteins were then subject to Western blot analysis with the purified rat anti-RPA antibody. (C) Interaction between GST-Xho and the purified Xenopus RPA in the presence or absence of DNase I. The proteins brought down by glutathione beads were analyzed by Western blot with the purified rat anti-RPA antibody. (D) Amount of RPA bound to GST-Stu/Xho. GST-Stu/Xho (500 nM) was incubated with 5 μl of cytosol in a 15-μl reaction and then brought down by glutathione beads. Indicated amounts (as percentages of cytosol) of the beads and supernatant fractions were analyzed by Western blot with the purified rat anti-RPA antibody.
Figure 7. Effect of RPA and gp32 on the helicase activity of FFA-1. (A) Helicase reactions containing the indicated amounts of FFA-1 and RPA (in nM). (B) Helicase reactions containing the indicated amounts of FFA-1 (in nM) and gp32 (in μM). In both A and B, the second lanes from the right contain buffer only, whereas the rightmost lanes contain the heated DNA substrate. All the reactions also contain 2.5 μM (in nucleotides) DNA and 2 mM ATP. Indicated on the left of each gel are the migration positions of double-stranded DNA markers.
Figure 8. Effect of GSTâFFA-1 fusion proteins on FFA-1 helicase activity. (A) In the presence of RPA. Lanes 1â10 contain 2 nM FFA-1 and 60 nM RPA, but lanes 1â9 also contain the various fusion proteins at the indicated final concentrations (in μM). Lane 11 contains the substrate incubated in buffer only and lane 12 contains the heated substrate. (B) In the absence of RPA. Lanes 1â3 contain 5 nM FFA-1 and GST-Xho (0.75 μM), GST-Stu/Xho (0.75 μM), and GST-Stu (1.5 μM). Lane 4 contains 5 nM FFA-1 only, lane 5 contains the substrate incubated in buffer only, and lane 6 contains the heated substrate. More FFA-1 was used in this experiment to better detect the weak helicase activity in the absence of RPA. The film was also exposed four times as long as that of A. All the reactions contain 2.5 μM DNA and 2 mM ATP.
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