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Evidence for a replication function of FFA-1, the Xenopus orthologue of Werner syndrome protein.

Chen CY, Graham J, Yan H - J. Cell Biol. (2001)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.

ABSTRACT
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.

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Colocalization of FFA-1 and sites of DNA synthesis. (A–F) In normal reconstituted nuclei. Biotin-dCTP was added 5 min before fixation. (A–C) 45 min. (D–F) 60 min. (G–L) In reconstituted nuclei formed in the presence of aphidicolin. (G–I) An aphidicolin-arrested nucleus directly fixed and stained. Biotin-dCTP was added to the reaction at the beginning. (H–L) 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.
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Figure 2: Colocalization of FFA-1 and sites of DNA synthesis. (A–F) In normal reconstituted nuclei. Biotin-dCTP was added 5 min before fixation. (A–C) 45 min. (D–F) 60 min. (G–L) In reconstituted nuclei formed in the presence of aphidicolin. (G–I) An aphidicolin-arrested nucleus directly fixed and stained. Biotin-dCTP was added to the reaction at the beginning. (H–L) 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.

Mentions: We first used indirect immunofluorescence staining to establish the relationship among FFA-1, RPA, and sites of DNA replication in nuclei reconstituted in Xenopus egg extracts. To do this, nuclei were reconstituted by mixing demembranated sperm chromatin and egg extracts. After various lengths of time they were fixed and then stained for FFA-1, RPA, and DNA synthesis. Replication began asynchronously, but generally occurred between 45 and 85 min in most nuclei. It was monitored by the incorporation of biotin-dCTP which was added to the reaction 5 min before fixation. As shown in Fig. 1, FFA-1 and RPA displayed an almost identical spatial distribution throughout DNA replication. During the early stage (Fig. 1, A–D; 40–45 min), they formed discrete foci on chromatin. Replication was not detectable in most nuclei, which is consistent with the observation that focus structure is formed before DNA synthesis. But when replication was detected, it occurred at a subset of FFA-1 foci (Fig. 2, A–C), suggesting that FFA-1 foci are indeed where replication is initiated. During the middle stage, both FFA-1 and RPA showed a more diffuse staining colocalizing with chromatin (Fig. 1, E–H; 45–85 min). Replication proceeded rapidly and was also detected throughout chromatin (Fig. 2, D–F). The diffuse FFA-1 and RPA staining subsided by the end of this stage and FFA-1 and RPA could again be detected as foci which often showed incorporation of biotin-dCTP (data not shown). During the late stage (after 85 min), replication was no longer detectable (data not shown), the diffuse staining of FFA-1 and RPA further decreased, but more discrete foci containing FFA-1 and RPA appeared again (Fig. 1, I–L) and persisted up to at least 150 min (the maximum time assayed).


Evidence for a replication function of FFA-1, the Xenopus orthologue of Werner syndrome protein.

Chen CY, Graham J, Yan H - J. Cell Biol. (2001)

Colocalization of FFA-1 and sites of DNA synthesis. (A–F) In normal reconstituted nuclei. Biotin-dCTP was added 5 min before fixation. (A–C) 45 min. (D–F) 60 min. (G–L) In reconstituted nuclei formed in the presence of aphidicolin. (G–I) An aphidicolin-arrested nucleus directly fixed and stained. Biotin-dCTP was added to the reaction at the beginning. (H–L) 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.
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Related In: Results  -  Collection

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Figure 2: Colocalization of FFA-1 and sites of DNA synthesis. (A–F) In normal reconstituted nuclei. Biotin-dCTP was added 5 min before fixation. (A–C) 45 min. (D–F) 60 min. (G–L) In reconstituted nuclei formed in the presence of aphidicolin. (G–I) An aphidicolin-arrested nucleus directly fixed and stained. Biotin-dCTP was added to the reaction at the beginning. (H–L) 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.
Mentions: We first used indirect immunofluorescence staining to establish the relationship among FFA-1, RPA, and sites of DNA replication in nuclei reconstituted in Xenopus egg extracts. To do this, nuclei were reconstituted by mixing demembranated sperm chromatin and egg extracts. After various lengths of time they were fixed and then stained for FFA-1, RPA, and DNA synthesis. Replication began asynchronously, but generally occurred between 45 and 85 min in most nuclei. It was monitored by the incorporation of biotin-dCTP which was added to the reaction 5 min before fixation. As shown in Fig. 1, FFA-1 and RPA displayed an almost identical spatial distribution throughout DNA replication. During the early stage (Fig. 1, A–D; 40–45 min), they formed discrete foci on chromatin. Replication was not detectable in most nuclei, which is consistent with the observation that focus structure is formed before DNA synthesis. But when replication was detected, it occurred at a subset of FFA-1 foci (Fig. 2, A–C), suggesting that FFA-1 foci are indeed where replication is initiated. During the middle stage, both FFA-1 and RPA showed a more diffuse staining colocalizing with chromatin (Fig. 1, E–H; 45–85 min). Replication proceeded rapidly and was also detected throughout chromatin (Fig. 2, D–F). The diffuse FFA-1 and RPA staining subsided by the end of this stage and FFA-1 and RPA could again be detected as foci which often showed incorporation of biotin-dCTP (data not shown). During the late stage (after 85 min), replication was no longer detectable (data not shown), the diffuse staining of FFA-1 and RPA further decreased, but more discrete foci containing FFA-1 and RPA appeared again (Fig. 1, I–L) and persisted up to at least 150 min (the maximum time assayed).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus