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. 2008 Oct 24;322(5901):597-602.
doi: 10.1126/science.1162790.

Functional targeting of DNA damage to a nuclear pore-associated SUMO-dependent ubiquitin ligase

Shigeki Nagai  1 , Karine Dubrana, Monika Tsai-Pflugfelder, Marta B Davidson, Tania M Roberts, Grant W Brown, Elisa Varela, Florence Hediger, Susan M Gasser, Nevan J Krogan
Affiliations

Functional targeting of DNA damage to a nuclear pore-associated SUMO-dependent ubiquitin ligase

Shigeki Nagai et al. Science. .

Abstract

Recent findings suggest important roles for nuclear organization in gene expression. In contrast, little is known about how nuclear organization contributes to genome stability. Epistasis analysis (E-MAP) using DNA repair factors in yeast indicated a functional relationship between a nuclear pore subcomplex and Slx5/Slx8, a small ubiquitin-like modifier (SUMO)-dependent ubiquitin ligase, which we show physically interact. Real-time imaging and chromatin immunoprecipitation confirmed stable recruitment of damaged DNA to nuclear pores. Relocation required the Nup84 complex and Mec1/Tel1 kinases. Spontaneous gene conversion can be enhanced in a Slx8- and Nup84-dependent manner by tethering donor sites at the nuclear periphery. This suggests that strand breaks are shunted to nuclear pores for a repair pathway controlled by a conserved SUMO-dependent E3 ligase.

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Figures

Fig. 1
Fig. 1
Relocation of an irreparable DSB to the nuclear periphery. (A) Galactose induces a DSB at MAT in a haploid strain that lacks homologous donor loci (11), bears lacO sites 4.4 kb from MATα, and expresses GFP-lacI and Nup49-GFP fusion proteins (GA-1496). (B) Position of the GFP-tagged MAT locus was scored relative to the NE (single-plane confocal images detecting Nup49 and MAT signals). Ratios of distance from NE and diameter in focal plane are binned into equal concentric zones (12). (C) Results of scoring MAT position in GA-1496 or in strains with either an uncleavable HO site (mataho, GA-1965) or HM loci (HML HMR; GA-2269). Red bar indicates random distribution, and an asterisk indicates significantly nonrandom. Number of cells analyzed and confidence values for a proportional test between random and experimental distribution are (for MATα and 2-hours glucose, 356 and P = 1.6 × 10−1; for mataho 2-hours galactose, 258 and P = 8.9 × 10−1; MATα 2-hours galactose, 195 and P = 4.5 × 10−8; and MATα+HM, 363 and P = 3.2 × 10−1). (D) 250 sequential confocal frames at 1.5-s intervals were aligned by the pore signals for G1 phase cells of the strains used in (C) (12), and MAT position was marked in each frame. Five-min trajectories are green. Bar indicates 1 µm.
Fig. 2
Fig. 2
Collapsed replication forks colocalize with pores. (A) Position of LacO-tagged early origin ARS607 (GA-1461) or late origin ARS1412 (GA-2070) was determined as in Fig. 1. (B) Exponentially growing GA-1461 cells were incubated ±0.2 M HU 1 or 2 hours. Spot position in S-phase cells was scored as in Fig. 1. Zone 1 enrichment by 2 hours is significant (P = 8 × 10−5). (C) Strains as in (A) were synchronized in G1 by α-factor treatment and released into 0.2 M HU ± 0.033% MMS for 1 hour. Zone 1 enrichment of collapsed forks is significant (asterisk indicates P = 2 × 10−5). (D) GFP-tagged ARS607 (red) and a CFP-Nup49 fusion (green) were expressed in a nup133Δ background expressing nup133Δ 44–236 (19). Cells synchronized in G1 phase were released 1 hour in 0.033% MMS plus 0.2 M HU. Deconvolved confocal sections of nuclei were scored for complete GFP-CFP signal overlap.
Fig. 3
Fig. 3
Nup84 complex and Slx5/Slx8 interact. (A) Hierarchical clustering of the nuclear function E-MAP (21). Genetic interactions from this map common to nup60Δ, nup84Δ, nup133Δ, slx5Δ, and slx8Δ are highlighted in blue and yellow. (B) Plot of correlation coefficients generated from comparison of the genetic profiles from slx5Δ or slx8Δ to all other profiles in this E-MAP. (C) Rat anti-HA (in green) and mouse anti–nuclear pore (Mab414, in red) colocalized by deconvolved confocal imaging of cells bearing Slx5-HA (GA-3867) or Slx8-HA (GA-3868). (D) Coimmunoprecipitation of Slx8 with Nup84 from cells bearing both Slx8-HA and Nup84-Myc (GA-5161) or Slx8-HA alone (GA-3868) (10). Westerns of proteins recovered with anti-Myc– or anti-HA–coated magnetic beads were probed with both antibodies. IgG, immunoglobulin G.
Fig. 4
Fig. 4
An irreparable DSB interacts with nuclear pores. (A and B) ChIP with anti-Myc in JKM179 derivatives with or without Nup84-Myc (GA-4133) or Nup133-Myc, with or without GAL1:HO (GA-4135 and GA-4140) at indicated time points on galactose. Primer/probe sets for real-time polymerase chain reaction are 0.6 kb, 1.6 kb, 4.5 kb, and 9.6 kb from the HO cut. For calculation of absolute enrichment over a mitochondrial gene, OLI1, see (10). (C) Anti–Nup84-Myc ChIP in a nup120Δ mutant (GA-4861) and a wild-type strain, as in (A). (D) Anti-Myc ChIP for Slx8-13Myc in GA-4137, as in (A). (E) ChIP for Nup84-Myc was performed in mec1Δsml1Δ (GA-4847), mec1Δtel1Δ sml1Δ, or wild-type strains. Absolute enrichment of MAT+1.6 kb over OLI1 as in (A). (F) The position of cleaved MAT was scored as in Fig. 1B in isogenic strains ± 2 hour HO induction. Strains and n and P values are as follows: WT (GA-1496) no cut: 365, 1.6 × 10−1; cut: 195, 4.5 × 10−8; yku70 (GA-1954) no cut: 118, 6.8 × 10−2; cut: 117, 2.6 × 10−11; mlp1mlp2 (GA-2228) no cut: 174, 3.7 × 10−2; cut: 184, 5.5 × 10−10: 148, 5.6 × 10−1; mec1-kd1 sml1 (13) (GA-2488) no cut: 269, 5.8 × 10−2; cut: 228, 9.2 × 10−2; sml1(13) (GA-2490) no cut: 205, 6.2 × 10−1, cut: 151, 1.7 × 10−8; rad9 (GA-580) no cut: 315, 2.3 × 10−1; cut: 309, 3.2 × 10−11.
Fig. 5
Fig. 5
Slx5/Slx8-Nup84 complex mutants affect recombination and GC rates. (A) Percentage cells (n > 200) containing Ddc2–yellow fluorescent protein (YFP) foci in strains carrying indicated gene deletions; averaging and standard deviation over three experiments. (B) GCR rates (18) are enhanced in nup84Δ and slx5Δ or slx8Δ strains; averaging and standard deviations from three fluctuation tests. (C) Recombination substrates for GC assays on nonhomologous chromosomes were modified by integrating four LexA sites upstream of the lys2 frameshift allele (white star;4lexA:lys2). ChrV carries 3′ truncated lys2 allele. GC renders cells Lys+. (D) Perinuclear anchoring of 4lexA: lys2 is achieved by expression of fusions LexA-Yif1 or LexA-Nup84 (fig. S7). Spontaneous GC rates in wild-type or indicated mutants as in (30) and (10). Double asterisks indicate suppression of slx8Δ-enhanced GC rate by LexA-Slx8 expression. (E) Rates of GC in (D) were normalized to +LexA values. (F) Model of Mec1/Tel1-dependent relocation of damage to Nup84/Slx5/Slx8 complexes that may ubiquinylate a substrate for proteolysis, enabling fork-associated repair.
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