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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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Effect of GST–FFA-1 fusion proteins on FFA-1 helicase activity. (A) In the presence of RPA. Lanes 1–10 contain 2 nM FFA-1 and 60 nM RPA, but lanes 1–9 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 1–3 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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Figure 8: Effect of GST–FFA-1 fusion proteins on FFA-1 helicase activity. (A) In the presence of RPA. Lanes 1–10 contain 2 nM FFA-1 and 60 nM RPA, but lanes 1–9 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 1–3 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.

Mentions: Since GST-Xho and GST-Stu/Xho can interact with RPA, we then addressed whether they can inhibit the stimulation of FFA-1 helicase activity by RPA. To do this, various amounts of the fusion proteins were added to the helicase reaction containing FFA-1 and RPA. As shown in Fig. 8 A, GST-Xho and GST-Stu/Xho inhibited the unwinding reaction in a dose-dependent manner. In contrast, GST-Stu had no effect on unwinding, even at the highest concentration (2.4 μM). In the absence of RPA, FFA-1 had a low intrinsic helicase activity, particularly on small fragments. But as shown in Fig. 8 B, this activity was not significantly affected by the fusion proteins. In addition, the affinity of RPA for single-stranded DNA was not affected by the fusion proteins (data not shown). Collectively, these results support the interpretation that RPA stimulates FFA-1 through direct protein–protein interaction and this stimulation is blocked by the two fusion proteins containing the Stu/Xho domain.


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)

Effect of GST–FFA-1 fusion proteins on FFA-1 helicase activity. (A) In the presence of RPA. Lanes 1–10 contain 2 nM FFA-1 and 60 nM RPA, but lanes 1–9 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 1–3 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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Related In: Results  -  Collection

Show All Figures
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Figure 8: Effect of GST–FFA-1 fusion proteins on FFA-1 helicase activity. (A) In the presence of RPA. Lanes 1–10 contain 2 nM FFA-1 and 60 nM RPA, but lanes 1–9 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 1–3 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.
Mentions: Since GST-Xho and GST-Stu/Xho can interact with RPA, we then addressed whether they can inhibit the stimulation of FFA-1 helicase activity by RPA. To do this, various amounts of the fusion proteins were added to the helicase reaction containing FFA-1 and RPA. As shown in Fig. 8 A, GST-Xho and GST-Stu/Xho inhibited the unwinding reaction in a dose-dependent manner. In contrast, GST-Stu had no effect on unwinding, even at the highest concentration (2.4 μM). In the absence of RPA, FFA-1 had a low intrinsic helicase activity, particularly on small fragments. But as shown in Fig. 8 B, this activity was not significantly affected by the fusion proteins. In addition, the affinity of RPA for single-stranded DNA was not affected by the fusion proteins (data not shown). Collectively, these results support the interpretation that RPA stimulates FFA-1 through direct protein–protein interaction and this stimulation is blocked by the two fusion proteins containing the Stu/Xho domain.

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