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Human Rif1 protein binds aberrant telomeres and aligns along anaphase midzone microtubules.

Xu L, Blackburn EH - J. Cell Biol. (2004)

Bottom Line: The hRif1 level rose during late S/G2 but hRif1 was not visible on chromosomes in metaphase and anaphase; however, notably, specifically during early anaphase, hRif1 aligned along a subset of the midzone microtubules between the separating chromosomes.In telophase, hRif1 localized to chromosomes, and in interphase, it was intranuclear.These results define a novel subcellular localization behavior for hRif1 during the cell cycle.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143, USA.

ABSTRACT
We identified and characterized a human orthologue of Rif1 protein, which in budding yeast interacts in vivo with the major duplex telomeric DNA binding protein Rap1p and negatively regulates telomere length. Depletion of hRif1 by RNA interference in human cancer cells impaired cell growth but had no detectable effect on telomere length, although hRif1 overexpression in S. cerevisiae interfered with telomere length control, in a manner specifically dependent on the presence of yeast Rif1p. No localization of hRif1 on normal human telomeres, or interaction with the human telomeric proteins TRF1, TRF2, or hRap1, was detectable. However, hRif1 efficiently translocated to telomerically located DNA damage foci in response to the synthesis of aberrant telomeres directed by mutant-template telomerase RNA. The hRif1 level rose during late S/G2 but hRif1 was not visible on chromosomes in metaphase and anaphase; however, notably, specifically during early anaphase, hRif1 aligned along a subset of the midzone microtubules between the separating chromosomes. In telophase, hRif1 localized to chromosomes, and in interphase, it was intranuclear. These results define a novel subcellular localization behavior for hRif1 during the cell cycle.

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Accumulation of hRif1 protein at DNA damage foci induced by MMS treatment. Images were acquired with a Deltavision microscopy system as described for Fig. 4. (A) LOX cells were treated with 0.01% MMS for 1 h, then fixed and immunostained with 53BP1 antibody (green) to visualize DNA damage foci, hRif1 antibody PAB2852 (red) to visualize the endogenous hRif1 protein, and DAPI (blue). (B) GFP-hRif1 fusion protein localizes to DNA damage foci induced by MMS treatment. LOX cells were transfected with pEGFP-hRif1 expression construct. 2 d after transfection, cells were treated with 0.01% MMS for 1 h, fixed and stained with 53BP1 antibody (red) and DAPI (blue). Bars, 10 μm.
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fig7: Accumulation of hRif1 protein at DNA damage foci induced by MMS treatment. Images were acquired with a Deltavision microscopy system as described for Fig. 4. (A) LOX cells were treated with 0.01% MMS for 1 h, then fixed and immunostained with 53BP1 antibody (green) to visualize DNA damage foci, hRif1 antibody PAB2852 (red) to visualize the endogenous hRif1 protein, and DAPI (blue). (B) GFP-hRif1 fusion protein localizes to DNA damage foci induced by MMS treatment. LOX cells were transfected with pEGFP-hRif1 expression construct. 2 d after transfection, cells were treated with 0.01% MMS for 1 h, fixed and stained with 53BP1 antibody (red) and DAPI (blue). Bars, 10 μm.

Mentions: It has recently been reported that hRif1 localizes to sites of general DNA damage (Silverman et al., 2004). To confirm that hRif1 plays a role in the general DNA damage response in the human cell lines used in this work, we induced DNA damage in LOX cells by treating the cells with MMS. Immunostaining of endogenous hRif1 protein after MMS treatment for 1 h revealed a punctate nuclear pattern for hRif1. Co-staining with antibody against 53BP1 confirmed that these hRif1 foci colocalized with 53BP1 (Fig. 7 A). The appearance and size of these hRif1 foci was reminiscent of the telomerically located foci that had been induced by the 47A mutant-template telomerase RNA expression as described above. To verify independently that the punctate nuclear pattern is specific to hRif1 protein, we also transfected LOX cells with a GFP-hRif1 fusion protein expression construct and then subjected the cells to the same 1-h MMS treatment. As shown in Fig. 7 B, GFP-hRif1 protein distributed heterogeneously in the nucleus in control cells, whereas after MMS treatment, GFP-hRif1 protein translocated to discrete foci that overlapped with 53BP1 protein. These data confirm that hRif1 plays a role in the response to general DNA damage. Hence, we suggest that the translocation of hRif1 to aberrant telomeres that we have observed reflects its role in a DNA damage response at uncapped telomeres.


Human Rif1 protein binds aberrant telomeres and aligns along anaphase midzone microtubules.

Xu L, Blackburn EH - J. Cell Biol. (2004)

Accumulation of hRif1 protein at DNA damage foci induced by MMS treatment. Images were acquired with a Deltavision microscopy system as described for Fig. 4. (A) LOX cells were treated with 0.01% MMS for 1 h, then fixed and immunostained with 53BP1 antibody (green) to visualize DNA damage foci, hRif1 antibody PAB2852 (red) to visualize the endogenous hRif1 protein, and DAPI (blue). (B) GFP-hRif1 fusion protein localizes to DNA damage foci induced by MMS treatment. LOX cells were transfected with pEGFP-hRif1 expression construct. 2 d after transfection, cells were treated with 0.01% MMS for 1 h, fixed and stained with 53BP1 antibody (red) and DAPI (blue). Bars, 10 μm.
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fig7: Accumulation of hRif1 protein at DNA damage foci induced by MMS treatment. Images were acquired with a Deltavision microscopy system as described for Fig. 4. (A) LOX cells were treated with 0.01% MMS for 1 h, then fixed and immunostained with 53BP1 antibody (green) to visualize DNA damage foci, hRif1 antibody PAB2852 (red) to visualize the endogenous hRif1 protein, and DAPI (blue). (B) GFP-hRif1 fusion protein localizes to DNA damage foci induced by MMS treatment. LOX cells were transfected with pEGFP-hRif1 expression construct. 2 d after transfection, cells were treated with 0.01% MMS for 1 h, fixed and stained with 53BP1 antibody (red) and DAPI (blue). Bars, 10 μm.
Mentions: It has recently been reported that hRif1 localizes to sites of general DNA damage (Silverman et al., 2004). To confirm that hRif1 plays a role in the general DNA damage response in the human cell lines used in this work, we induced DNA damage in LOX cells by treating the cells with MMS. Immunostaining of endogenous hRif1 protein after MMS treatment for 1 h revealed a punctate nuclear pattern for hRif1. Co-staining with antibody against 53BP1 confirmed that these hRif1 foci colocalized with 53BP1 (Fig. 7 A). The appearance and size of these hRif1 foci was reminiscent of the telomerically located foci that had been induced by the 47A mutant-template telomerase RNA expression as described above. To verify independently that the punctate nuclear pattern is specific to hRif1 protein, we also transfected LOX cells with a GFP-hRif1 fusion protein expression construct and then subjected the cells to the same 1-h MMS treatment. As shown in Fig. 7 B, GFP-hRif1 protein distributed heterogeneously in the nucleus in control cells, whereas after MMS treatment, GFP-hRif1 protein translocated to discrete foci that overlapped with 53BP1 protein. These data confirm that hRif1 plays a role in the response to general DNA damage. Hence, we suggest that the translocation of hRif1 to aberrant telomeres that we have observed reflects its role in a DNA damage response at uncapped telomeres.

Bottom Line: The hRif1 level rose during late S/G2 but hRif1 was not visible on chromosomes in metaphase and anaphase; however, notably, specifically during early anaphase, hRif1 aligned along a subset of the midzone microtubules between the separating chromosomes.In telophase, hRif1 localized to chromosomes, and in interphase, it was intranuclear.These results define a novel subcellular localization behavior for hRif1 during the cell cycle.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143, USA.

ABSTRACT
We identified and characterized a human orthologue of Rif1 protein, which in budding yeast interacts in vivo with the major duplex telomeric DNA binding protein Rap1p and negatively regulates telomere length. Depletion of hRif1 by RNA interference in human cancer cells impaired cell growth but had no detectable effect on telomere length, although hRif1 overexpression in S. cerevisiae interfered with telomere length control, in a manner specifically dependent on the presence of yeast Rif1p. No localization of hRif1 on normal human telomeres, or interaction with the human telomeric proteins TRF1, TRF2, or hRap1, was detectable. However, hRif1 efficiently translocated to telomerically located DNA damage foci in response to the synthesis of aberrant telomeres directed by mutant-template telomerase RNA. The hRif1 level rose during late S/G2 but hRif1 was not visible on chromosomes in metaphase and anaphase; however, notably, specifically during early anaphase, hRif1 aligned along a subset of the midzone microtubules between the separating chromosomes. In telophase, hRif1 localized to chromosomes, and in interphase, it was intranuclear. These results define a novel subcellular localization behavior for hRif1 during the cell cycle.

Show MeSH
Related in: MedlinePlus