The Nup84 complex affects survival to DSBs in subtelomeres

Having shown that telomere XI-L was delocalized in Nup84 complex mutants, we investigated the efficiency of survival and repair to DSBs in these mutant backgrounds. To generate DSBs, we expressed the I-SceI endonuclease in vivo in haploid strains carrying a URA3 marker flanked by two inverted I-SceI cutting sites at defined positions (Fig. 2 A and Ricchetti et al., 2003). Cell survival is monitored by comparing the number of colonies formed after plating on inducing (galactose) versus noninducing (glucose) conditions, whereas efficiency of DSB formation is followed by measuring the percentage of [URA^-] colonies after replica plating (see Materials and methods). In a wild-type haploid strain, most of the cells are unable to form colonies after induction of DSBs on galactose, but survival increases by a factor of 20 when the DSB is in subtelomeres (Ricchetti et al., 2003). We analyzed survival to both a cut 3.5 kb from the left telomere (L1 in Ricchetti et al., 2003) and a cut in a central region, 64 kb from the left telomere (C5) of chromosome XI in cells mutated in the Nup84 complex and in NUP170, a nucleoporin peripheral to this complex. As previously shown, when the DSB happens in a central region, cell survival is very low (Fig. 2 A and Table I ; Ricchetti et al., 2003). We found that cell survival is comparable between wild type, Δnup145C, Δnup84, and Δnup170 strains (Fig. 2 A and Table I). On the contrary, in the case of a subtelomeric DSB, whereas in a wild-type strain cell survival reaches ~4% (Fig. 2 A and Table I; Ricchetti et al., 2003), it decreases by 93% and 71% in Δnup145C and Δnup84 strains, respectively, and by 42% and 41% in Δnup120 and Δnup133, while it is only partially diminished in the Δnup170 strain (20%; Table I). Therefore, in all Nup84 complex mutants tested here a defect in cell survival after generation of a DSB was observed, with survival rates of cells bearing a DSB in a central and in subtelomeric region of the same chromosome becoming similar in Δnup145C and to a lesser extent in Δnup84 mutants, indicating that in these mutants the dependency of the survival rate on the location of the DSB along the chromosome is abolished.

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Figure 2.

Nup84 complex is required for subtelomeric DSB repair efficiencies. (A) Survival to DSBs according to their position in wild-type and different nucleoporin mutant backgrounds. Chromosomes XI and XV are schematized. Black dots and vertical hatched boxes represent centromere and subtelomeres, respectively. Arrows indicate the positions of induced DSBs generated by cleavage of two inverted I-SceI cutting sites (cs) surrounding the URA3 marker (black box). Size in kb is indicated. Colony forming units (CFU) are calculated as the ratio of galactose growing colonies (GAL) versus glucose growing colonies (GLU) expressed in percentages (%). Values obtained for wild-type (WT, white), Δnup145C (black), Δnup84 (gray), Δnup133 (diagonals), Δnup120 (horizontal lines), and Δnup170 (white dots) are shown according to DSB's position. Subtel. XI stands for subtelomeric XI-L. Standard errors with a P value of 0.1 are shown. (B) Proportion of repair by NHEJ and rearrangements in subtelomere XI-L. Subtelomeric DSB is schematized as in A. Percentages of repair either by NHEJ or by gross chromosomal rearrangements per plated cells are shown for wild-type (WT, white) and Δnup145C (black) (C). Repair machinery is functional in Δnup145C. Left; DSB is generated in a central part of chromosome XV and gene conversion is made possible because of homologous sequences present on chromosome V (gray box, see text and Karathanasis and Wilson, 2002). Colony forming units are calculated as in A. Middle; DSB is generated in the subtelomeric position in diploid wild-type cells or diploid cells homozygous for Δnup145C. Colony forming units are calculated as in A. Right; DSB is created on a replicative plasmid by restriction enzymes generating cohesive or blunt ended ends. Wild-type (WT) and Δnup145C strains were transformed with equivalent amounts of supercoiled or linearized pRS316 (ARS-CEN-URA3) plasmid. The number of transformants obtained with the linearized plasmid expressed as a percentage of the number of transformants obtained with the supercoil DNA are plotted. Experiments were repeated three times and error bars represent the 95% confidence intervals.

Ratios of different types of repair are unaffected in Nup84 complex mutants

The decrease in cell survival could reflect either a problem in repair efficiency or some checkpoint-related problem in response to DNA damage. The latter was excluded because the Δnup145C mutant adapts and recovers subtelomeric DSB like a wild-type strain, whereas the checkpoint Δrad17 mutant does not (see Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200505159/DC1; and Lee et al., 1998). We therefore analyzed the mechanisms by which the DSBs were repaired. 200 colonies that formed on galactose-containing medium were screened by PCR for possible repair by NHEJ. The absolute frequency of repair was calculated by scoring the number of positive PCRs relative to the number of plated cells (in noninducing conditions). In a wild-type strain, 0.21% (±0.039) of plated cells were repaired by NHEJ; this value remaining constant along the chromosome (Ricchetti et al., 2003; and our unpublished data). The absolute frequency of NHEJ drops to 0.014% (±0.002) in Δnup145C mutant cells when DSBs are generated close to the telomere, but not when the cut is central (Fig. 2 B and unpublished data). This shows that a component of the Nup84 complex is required for efficient repair by NHEJ in subtelomeres. However, when the frequency of repair events is calculated among the survivors, similar values are obtained in wild-type and Δnup145C mutant strains (respectively 29% ± 7 and 25% ± 7), indicating that when repair of the DSB allows colony formation, the ratio of NHEJ to other mechanisms is the same in both backgrounds. 21 of these subtelomeric NHEJ events that occurred in Δnup145C mutants were sequenced, and repair event corresponded in all cases to NHEJ involving only minor rearrangements (Fig. 3 ). The majority of the cases (60%) correspond to a ligation between the two cut sites with insertion of two nucleotides, as already observed in a wild-type context (type II in Fig. 3 and in Ricchetti et al., 2003). The remaining cases (40%) correspond to a major resection of both I-SceI cutting sites, which was not previously described (therefore named type VII in Fig. 3).

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Figure 3.

Sequences of the junction of NHEJ events at locus L1 in Δnup145C and proposed mechanisms of NHEJ. Coordinates of the L1 locus on chromosome XI-L are shown. I-SceI restriction sites (in opposite orientations) are in bold, cleavage position generating 4-bp 3′ overhangs is indicated by a staggered line, BamHI and KpnI restriction sites are boxed and plasmid sequences are in lower case letters (see Fairhead et al., 1996 for construction details). Possible end processing and subsequent alignment of complementary base pairs are shown. See text for details.

We then analyzed the nature of non-NHEJ events at the L1 locus. The absolute frequency of these events (i.e., the number of events calculated on the total number of plated cells) is 4% in the wild-type strain, and drops to 0.25% in the Δnup145C strain (Fig. 2 B). Comparison of the Southern blot patterns (as in Ricchetti et al., 2003) of repaired chromosomes in wild-type and Δnup145C cells shows only minor variations in the frequencies of different types of repair, and no novel pattern in the mutant (unpublished data).

The repair machinery is not affected in a Δnup145C mutant strain

The observation that DSB repair is efficient in NPC mutants when the DSB is generated in a central region of a chromosome argues against a defect in nuclear transport of enzymes involved in the repair pathways. Nonetheless, we designed several experiments to confirm this. First, we studied cell survival and DSB repair using a similar experimental setup, but in which expression of I-SceI endonuclease is driven from the gene, flanked by two I-SceI cutting sites, integrated on chromosome XV at the central ADE2 locus (Karathanasis and Wilson, 2002). In this system, the presence of an ade2-K1 mutated allele inserted into CAN1 (YEL063c) 31.7kb from telomere V-L allows visualization of GC events. In these strains, induction of I-SceI expression leads to loss of the URA3 marker in 95% of cells. As was observed for a DSB generated in a central domain of chromosome XI, cell survival is similar in both wild-type and Δnup145C strains (Fig. 2 A and Table I). Frequencies of repair either by NHEJ or GC are also unchanged, indicating that, in the Δnup145C strain, the repair machinery is functional and has access to a central chromatin domain (Fig. 2 C and Table I). We next asked how a subtelomeric DSB can be repaired when homologous sequences are present. We thus constructed a diploid strain homozygous for the Δnup145C deletion and heterozygous for the cassette inserted at the subtelomeric L1 position. Cell survival after induction of the DSB was high in both wild-type and homozygous Δnup145C (91.5% ± 6.9 and 84% ± 11.4) with a cutting efficiency of 100%. This indicates that the inability to repair a subtelomeric DSB observed in a haploid Δnup145C strain is overtaken in a diploid strain. Finally, we used an assay that relies on the in vivo repair of a linearized plasmid required for its propagation in yeast cells (Fig. 2 C; Teo and Jackson, 1997). Because the DSB is generated in a region that is not homologous to the yeast genome, repair operates only through NHEJ. We determined the number of transformants recovered after transformation with a linearized and purified ARS-CEN-URA3 plasmid (pRS316; Sikorski and Hieter, 1989) relative to an uncut control both in wild-type and Δnup145C strains (Fig. 2 C). Restrictions leaving either 5′ overhangs (HindIII) or blunt ends (SmaI) were chosen. No differences in efficiency recovery were observed in both strains for these two types of ends, although SmaI linearized plasmid was less efficiently repaired (WT 2.1% ± 0.7; Δnup145C 2.9% ± 0.2) than HindIII (WT 24.2% ± 7.8; Δnup145C 16.5 ± 7.8), as expected (Hegde and Klein, 2000). All the above results indicate that the repair machinery is functional in Δnup145C, and therefore deficiencies in subtelomeric repair cannot be explained by a general deficiency of repair in the mutant.

Nup84p complex affects subtelomeric silencing

Because repair of a subtelomeric DSB is affected when members of the Nup84 complex are mutated, we analyzed whether silencing of subtelomeric chromatin is affected in these mutants. XI-L subtelomere was shown to be one of the most efficiently silenced subtelomeres (Pryde and Louis, 1999). Transcriptional silencing of a URA3 marker integrated into this subtelomeric region is measured by counting the fraction of cells that are able to grow in the presence of 5-fluoro-orotic acid (5-FOA), a toxic analogue of uracil. Reverse growth of 5-FOA–resistant clones on medium lacking uracil verifies that resistance is due to subtelomeric silencing rather than mutation of the URA3 gene. Compared with the wild-type strain, disruption of Nup84 complex (i.e., Δnup145C, Δnup84, Δnup120, and Δnup133) reduces URA3 silencing with variable strength, leading to very little or partial cell growth on 5-FOA (Fig. 4 ). On the contrary, silencing of native subtelomere XI-L remains intact when other nucleoporin genes (i.e., Δnup170 and Δnup53) are disrupted. Furthermore, silencing was restored when the corresponding wild-type nucleoporin was used for complementation, as shown for NUP145C, NUP84, and NUP133 (Fig. 4). These results show that defects in telomere position effect (TPE) are specific for the Nup84 complex. In addition, transcomplementation of silencing defects by the nup133ΔN mutant, lacking the amino-terminal encoding part of Nup133p, which only affects NPC clustering (Doye et al., 1994), indicates that NPC distribution is not involved in the silencing defects observed for native telomere XI-L. We then asked whether defect in silencing corroborated with a delocalization of Sir3p. This was monitored by microscopy using a Sir3-GFP fusion protein; addition of the GFP moiety to the COOH terminus of Sir3p having no effect on TPE of native subtelomere XI-L (Fig. S2 A). In a wild-type strain Sir3-GFP localizes in few dots (4–6) located at the nuclear periphery (Fig. S2 B). In Δnup145C or Δnup84 mutants, a nucleoplasmic staining is visible and Sir3-GFP localizes to an increased number of foci (6–8), indicating a partial delocalization of Sir3-GFP in Nup84 complex mutants that may be responsible for the defect in TPE.

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Figure 4.

Mutants of the Nup84 complex are defective for subtelomeric silencing. (A) The native configuration of chromosome XI-L subtelomere is schematized. Black arrows represent TG1-3 repeats; gray box, the coreX sequence which includes the ACS sequence; white triangle, the position of URA3 insertion (see Pryde and Louis, 1999 for details). (B) Measurement of repression of URA3 inserted at ACS of coreX. 10-fold dilutions were spotted on rich medium (YPD), on synthetic complete medium lacking uracil (SC-URA), and on medium containing 5-FOA. On this last medium wild-type cells bearing URA3 inserted into ACS sequence of core X (URA3@X-ACS) are able to repress this gene as illustrated by their ability to grow on FOA-containing medium. On the contrary, minimal repression is seen in Nup84 complex mutants (Δnup145C, Δnup84, Δnup120, Δnup133). No effect on telomeric repression is seen in other mutants (Δnup170, Δnup53). Silencing defects are complemented by full-length (FL) or the carboxy-terminal encoding part (C) of corresponding wild-type gene; Δnup133 silencing defect are complemented both by the wild-type gene (FL) or the nup133ΔN separation-of-function mutant (ΔN) that clusters NPCs (see Doye et al., 1994 for details). (C) Quantification of silencing defects. Cells were grown in nonselective conditions and 10-fold dilutions were plated onto YPD and medium containing 5-FOA.

Fluorescence observation and QuIA analysis

Haploid cells tagged by tetO repeats on chromosome XI-L (see Table II) were grown to exponential phase at 25°C in 5ml of YPD. When concentration reached 1–2 × 10⁶ cells/ml, cells from 1 ml aliquots were washed in synthetic complete medium and resuspended into 500 μl of the same medium. 5 μl were spotted onto slides, covered by a thin layer of agar (1.5% agar in synthetic complete medium). Images were captured with an UltraView RS Nipkow-disk confocal system (Perkin-Elmer) and controlled by PE-viewer software. A 100× objective (Plan Apo, 1.4 NA, oil immersion; Carl Zeiss MicroImaging, Inc.) was used. The pixel size is 65.8 nm. Stacks with a slice spacing of 200 nm were taken using a laser excitation of 488 nm with an acquisition time of 200 ms each. An analysis of each projected stack was performed with QuIA software. This software relies on the quantitative detection of the labeled chromosomal locus via its 3D coordinates (X, Y, Z) in the nuclear space by a multi-resolution method using a 3D wavelet transformation, allowing to correlate information from different sub-bands of analysis and to select spot-like structures only within the nuclear volume (Olivo-Marin, 2002). The output of the detection step are the coordinates of the center of the chromosomal locus and of the nucleus, from which a first estimate of their relative distance is computed, before correction of Z distortion. TetR-GFP background allows for nucleoplasm detection and calculation of R_est, i.e., the mean of all radii on the median focal plane, assuming the nucleus is spherical. In wild-type cells, border of TetR-GFP background coincides with Nup49-GFP staining. Z-distortion and Z-detection artifacts are corrected thanks to the values obtained from localization of a GFP fusion with Spc29p, a single pole body (SPB) protein. In yeast, the SPB is embedded in the nuclear envelope. Without any Z distortion, the distance between the SPB and the center of nucleus, normalized by the R_est value (d_SPB/R_est), should be constant whatever the Z coordinate of the SPB. On the contrary, the observed distribution d_SPB/R_est increases with the distance along the Z-axis of the SPB from the median plane of the nucleus. This distribution is best fitted by the second-degree polynomial curve: d_SPB/R_est = 0.3309(Z_SPB − Z_{nucleus center})² + 0.0129(Z_SPB − Z_{nucleus center}) + 0.8457. This equation was used to determine the compensatory term to be removed from all the measured radial distances between the barycentre of tetO locus and of the nucleus (d_tetO) and normalized by the R_est value (dtetO/Rest). Thus the corrected ratio, termed relative 3D position (R3Dp) is d_tetO/R_est = d_tetO/R_est − (0.3309(Z_TetO − Z_{nucleus center})² + 0.0129(Z_tetO − Z_{nucleus center})). In each strain, percentages of cells according to their R3Dp are then either distributed in two zones of equal volume (i.e., 50% of the volume each, the outmost external one defined as the peripheral one) or presented as a cumulative distribution function (CDF), allowing for a Kolmogorov-Smirnov test that determines the statistical differences between two distributions. Note that the depth of the most external volume of a sphere divided in two equal volumes is similar to the depth of the most external area of a circular surface divided in three equal areas (see Hediger et al., 2002b). Analyses were performed on at least 200 nuclei in at least three independent experiments. The average nuclear radius in mutant cells versus WT cells was measured and shown to be similar between strains (0.97 μm ± 0.02, in WT and Δesc1; 0.98 μm ± 0.03 in Δsir4 and Δnup133; 0.99 ± 0.11 in Δnup145C; 1.02 ± 0.04 in Δku70; 1.07 ± 0.04 in Δku70 Δnup145; 0.92 ± 0.02 in Δnup120 and 1.15 ± 0.13 in Δnup84). In Δnup84 mutant, cell morphology is more variable, but distorted cells do not exceed 10% of the entire population.

Silencing assays

Silencing of a URA3 marker inserted at the core X of chromosome XI-L (Pryde and Louis, 1999) was scored after growth of strains in nonselective conditions (YPD) to mid-log phase. 10-fold serial dilutions were made and spotted or plated to either SC-URA or plates containing 5-FOA. The percentage of cells giving rise to 5-FOA resistant colonies was calculated after 2–3 d of growth.

Induction of I-SceI DSBs in vivo

Wild-type, Δnup145C, Δnup84, Δnup133, Δnup120, Δnup170, or Δesc1 strains containing the URA3 cassette flanked by two inverted I-SceI cutting sites at different positions along chromosome XI were transformed by the I-SceI expression plasmids, either pPEX7 (Fairhead and Dujon, 1993) or pPEX14 (this paper). Both plasmids carry the I-SceI ORF controlled by a galactose-inducible promoter and a leu2d marker. pPEX14 corresponds to a version of pPEX7 in which a TRP1 marker is inserted, allowing for efficient selection of transformants on SC-TRP. Induction of DSB was performed as in Ricchetti et al. (2003). Experiments were repeated at least five times with at least two independent pPEX transformants each time.

PCRs and genomic analyses

Analyses of repair events at the molecular level were done according to Ricchetti et al. (2003). The frequency of repair events was calculated as the ratio of the number of these events divided by the number of colonies growing on galactose. The absolute frequencies were determined in each strain by dividing the previous value by the number of plated cells on glucose medium.

We greatly thank Pascal Roux and the Dynamic Imagery Platform (PFID) of the Pasteur Institute for help with confocal microscopy; Hervé Waxin for help with pulse field gel electrophoresis; Valérie Doye (Institut Curie, Paris, France), Ed Louis (University of Leicester, Leicester, UK), Kym Nasmyth, and Tom Wilson (University of Michigan Medical School, Ann Arbor, MI) for strains and plasmids; Frank Feuerbach and Olivier Gadal (Institut Pasteur, Paris, France) for sharing strains, oligonucleotides, and plasmids and for improving the manuscript by their constructive criticisms; Miria Ricchetti and Bertrand Llorente for critical reading; Christophe Zimmer, Henri Buc, and Jim Haber for their enlightening discussions; and all members of our labs for sharing time and discussions.

P. Therizols and G. Cabal are recipients of predoctoral fellowships of the Ministère de la Recherche through the Université Paris 6 and Paris 11, respectively. A. Genovesio was supported by fellowships from the Agence Nationale de Recherches sur le Sida (ANRS), Centre National de la Recherche Scientifique, and Institut Pasteur. This work benefited from ARC grant 3266. B. Dujon is member of the Institut Universitaire de France.