Conserved interactions of the splicing factor Ntr1/Spp382 with proteins involved in DNA double-strand break repair and telomere metabolism

The ligation of DNA double-strand breaks in the process of non-homologous end-joining (NHEJ) is accomplished by a heterodimeric enzyme complex consisting of DNA ligase IV and an associated non-catalytic factor. This DNA ligase also accounts for the fatal joining of unprotected telomere ends. Hence, its activity must be tightly controlled. Here, we describe interactions of the DNA ligase IV-associated proteins Lif1p and XRCC4 of yeast and human with the putatively orthologous G-patch proteins Ntr1p/Spp382p and NTR1/TFIP11 that have recently been implicated in mRNA splicing. These conserved interactions occupy the DNA ligase IV-binding sites of Lif1p and XRCC4, thus preventing the formation of an active enzyme complex. Consistently, an excess of Ntr1p in yeast reduces NHEJ efficiency in a plasmid ligation assay as well as in a chromosomal double-strand break repair (DSBR) assay. Both yeast and human NTR1 also interact with PinX1, another G-patch protein that has dual functions in the regulation of telomerase activity and telomere stability, and in RNA processing. Like PinX1, NTR1 localizes to telomeres and associates with nucleoli in yeast and human cells, suggesting a function in localized control of DSBR

Ntr1p overabundance affects NHEJ of linearized plasmid DNA transformed into yeast, or of chromosomal DNA double-strand breaks in different genetic backgrounds

<p><b>Copyright information:</b></p><p>Taken from "Conserved interactions of the splicing factor Ntr1/Spp382 with proteins involved in DNA double-strand break repair and telomere metabolism"</p><p></p><p>Nucleic Acids Research 2007;35(7):2321-2332.</p><p>Published online 27 Mar 2007</p><p>PMCID:PMC1874655.</p><p>© 2007 The Author(s)</p> () Wild-type (wt) or NHEJ-deficient Δ yeast strains constitutively producing full-length EGFP-tagged-Ntr1p from plasmid pUG36 (vector control) were transformed with equal amounts of digested or undigested plasmid pBTM116, which is a substrate for NHEJ (). Results are presented as relative transformation efficiencies (ratios of cut:uncut plasmid). EcoRI cut indicates 5′-overhangs, PstI cut indicates 3′-overhangs. Error bars represent one standard deviation, statistical significance (-values) by a two-tailed students -test is indicated. () Wild-type (wt), HR-deficient Δ, or NHEJ-deficient Δ strains constitutively producing full-length Ntr1p from plasmid pAS2–1. Chromosomal breaks were induced by additional expression of EcoRI or HO (GAL1-inducible) upon transformation of the respective expression vectors (). Results are presented as percentage of survival of cells carrying an NTR1-expressing vector or the respective control vector (pAS2-1) when grown on galactose containing medium. Error bars represent one standard deviation, -values of a two-tailed students -test are indicated

Yeast and human NTR1 co-localize with nucleolus- and telomere-associated proteins

<p><b>Copyright information:</b></p><p>Taken from "Conserved interactions of the splicing factor Ntr1/Spp382 with proteins involved in DNA double-strand break repair and telomere metabolism"</p><p></p><p>Nucleic Acids Research 2007;35(7):2321-2332.</p><p>Published online 27 Mar 2007</p><p>PMCID:PMC1874655.</p><p>© 2007 The Author(s)</p> () Intracellular localization of Ntr1p and co-localization with other proteins. Upper panel: EGFP-Ntr1p (green) localizes to the nucleus (DAPI, light blue) and forms foci. Middle panel: live cell images of CFP-Nop1p (red, false color) and EGFP-Ntr1p (green). Co-localizing signals are shown in yellow in the merge panel. Lower panel: confocal images of EGFP-Ntr1p (green) in fixed cells immunostained for Rap1p (red). DAPI staining of DNA is shown in blue. Co-localization between the two proteins is shown in yellow on the merge panel. () Intracellular localization of human NTR1 and TRF1. WI26 VA4 cells were co-transfected with expression constructs of ECFP-NTR1 (amino acids 289–580) and RFP-TRF1 (upper three panels). eCFP was used as a control (lower panel). Co-localization in confocal images is shown in yellow on the merge panel and telomeric co-localization is indicated by arrows