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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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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 anti–FFA-1C (top) and rabbit anti-RPA (bottom). (B) Mapping of the RPA interaction domain in FFA-1. The various GST–FFA-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.
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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 anti–FFA-1C (top) and rabbit anti-RPA (bottom). (B) Mapping of the RPA interaction domain in FFA-1. The various GST–FFA-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.

Mentions: We then studied the biochemical mechanism by which the dominant negative fusion proteins inhibit DNA synthesis at “hybrid foci.” The simplest explanation is that the fusion proteins displace the endogenous FFA-1 and the redundant protein from “hybrid foci.” However, since the endogenous FFA-1 is still present at “hybrid foci,” this explanation is unlikely to be the case. The second explanation is that, since helicases usually act as oligomers, the dominant negative fusion proteins, which do not contain the helicase domain, might interact with the endogenous FFA-1, leading to the formation of inactive helicase oligomers. We have tested this idea, but found no interaction between the fusion proteins and FFA-1 (data not shown); as such it is also unlikely to be true. The third explanation is based on the report that the helicase activity of human WRN can be stimulated by the human RPA protein, probably by protein–protein interaction (Shen et al. 1998b; Brosh et al. 1999). Conceivably, the helicase activity of FFA-1 may also be stimulated by RPA through protein–protein interaction. If the dominant negative fusion proteins can also interact with RPA, they may then interfere with this stimulation. To test this idea, we first determined whether FFA-1 could indeed interact with RPA by coimmunoprecipitation. Protein A beads were coated with different antibodies and then incubated with the cytosol. The proteins bound to the beads were then separated on SDS-PAGE, transferred to PVDF membranes, and probed with either anti–FFA-1 or anti-RPA antibodies. As shown in Fig. 6 A, the anti–FFA-1 antibody brought down not only FFA-1, but also a small amount of RPA. Conversely, the anti-RPA antibody brought down RPA and a small amount of FFA-1. In neither case did the control antibody bring down FFA-1 and RPA. This experiment suggests that FFA-1 and RPA can physically interact with each other.


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)

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 anti–FFA-1C (top) and rabbit anti-RPA (bottom). (B) Mapping of the RPA interaction domain in FFA-1. The various GST–FFA-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.
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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 anti–FFA-1C (top) and rabbit anti-RPA (bottom). (B) Mapping of the RPA interaction domain in FFA-1. The various GST–FFA-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.
Mentions: We then studied the biochemical mechanism by which the dominant negative fusion proteins inhibit DNA synthesis at “hybrid foci.” The simplest explanation is that the fusion proteins displace the endogenous FFA-1 and the redundant protein from “hybrid foci.” However, since the endogenous FFA-1 is still present at “hybrid foci,” this explanation is unlikely to be the case. The second explanation is that, since helicases usually act as oligomers, the dominant negative fusion proteins, which do not contain the helicase domain, might interact with the endogenous FFA-1, leading to the formation of inactive helicase oligomers. We have tested this idea, but found no interaction between the fusion proteins and FFA-1 (data not shown); as such it is also unlikely to be true. The third explanation is based on the report that the helicase activity of human WRN can be stimulated by the human RPA protein, probably by protein–protein interaction (Shen et al. 1998b; Brosh et al. 1999). Conceivably, the helicase activity of FFA-1 may also be stimulated by RPA through protein–protein interaction. If the dominant negative fusion proteins can also interact with RPA, they may then interfere with this stimulation. To test this idea, we first determined whether FFA-1 could indeed interact with RPA by coimmunoprecipitation. Protein A beads were coated with different antibodies and then incubated with the cytosol. The proteins bound to the beads were then separated on SDS-PAGE, transferred to PVDF membranes, and probed with either anti–FFA-1 or anti-RPA antibodies. As shown in Fig. 6 A, the anti–FFA-1 antibody brought down not only FFA-1, but also a small amount of RPA. Conversely, the anti-RPA antibody brought down RPA and a small amount of FFA-1. In neither case did the control antibody bring down FFA-1 and RPA. This experiment suggests that FFA-1 and RPA can physically interact with each other.

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