A moonlighting metabolic protein influences repair at DNA double-stranded breaks

Abstract

Catalytically active proteins with divergent dual functions are often described as 'moonlighting'. In this work we characterize a new, chromatin-based function of Lys20, a moonlighting protein that is well known for its role in metabolism. Lys20 was initially described as homocitrate synthase (HCS), the first enzyme in the lysine biosynthetic pathway in yeast. Its nuclear localization led to the discovery of a key role for Lys20 in DNA damage repair through its interaction with the MYST family histone acetyltransferase Esa1. Overexpression of Lys20 promotes suppression of DNA damage sensitivity of esa1 mutants. In this work, by taking advantage of LYS20 mutants that are active in repair but not in lysine biosynthesis, the mechanism of suppression of esa1 was characterized. First we analyzed the chromatin landscape of esa1 cells, finding impaired histone acetylation and eviction. Lys20 was recruited to sites of DNA damage, and its overexpression promoted enhanced recruitment of the INO80 remodeling complex to restore normal histone eviction at the damage sites. This study improves understanding of the evolutionary, structural and biological relevance of independent activities in a moonlighting protein and links metabolism to DNA damage repair.

Figure 1.

The Lys20 moonlighting function localized to a 20 amino acid C-terminal domain. (A) Ribbon diagram of an HCS homodimer depicting Lys4, the Schizosaccharomyces pombe homolog of Lys20 (44). The monomers are distinguished by coloring: violet and maroon. (B) Ribbon diagram of a Lys20 monomer. The S. cerevisiae Lys20 structure was modeled with the PyMOL Molecular Graphics System, using the crystal structure of Lys4 of S. pombe as a template. The lid domain is represented in green. (C) Localization of the residues evaluated in the structure-function assays that identified the moonlighting domain of Lys20. The localization of the color-coded residues in a linear cartoon of the protein is shown below. E155 is a previously characterized catalytic amino acid of Lys20. Of the three amino acids necessary for metal binding (E32, H212 and H214), H212 and H214 were tested. GIGERNG is a highly conserved domain that is found close to the catalytic region. Because the crystal structure of the template Lys4 terminated at A391, the moonlighting domain and the NLS of Lys20 could not be modeled. (D) Residues I313, L314 and V399 to I418 were not necessary for the biosynthetic activity of Lys20. Growth assays of lys20Δ lys21Δ strains transformed with LYS20 mutants. Growth on medium without lysine indicated that the strain was proficient for HCS catalytic activity. (E) Residues V399 to I418 encompassed the moonlighting domain of Lys20. Growth assay of esa1–414 strains plated on medium with the DSB-inducing drug camptothecin (CPT) or on the vehicle control DMSO. Suppression of the DNA damage sensitivity of esa1–414 by increased LYS20 gene dosage was used as a control to analyze the ability of lys20 mutants in (D) to suppress esa1–414. The lys20-V399-I418Δ (lys20-moon) mutant was unable to suppress esa1–414 DNA damage sensitivity, yet remained proficient for lysine synthesis.

Figure 2.

LYS20 interacted genetically with DNA damage response genes. DNA damage sensitive strains were transformed with vector or with the lys20-cat mutant (lys20-E155A) that was competent for moonlighting activity but not for lysine biosynthesis. Increased dosage of lys20-cat improved the DNA damage sensitivity of esa1–414 and tel1Δ, but interfered in rad6Δ cells. The lys20-cat mutant was used because it was more proficient in suppressing esa1–414 compared to wild-type LYS20 (Figure 1E).

Figure 3.

Lys20 was recruited to DNA DSBs with similar kinetics to the HAT Esa1. (A) Increased lys20-cat dosage did not promote increased global histone H4 acetylation. Protein lysates were probed as indicated. Tubulin was included as a loading control. (B) Subcellular fractionation assays revealed that Lys20 binding to chromatin was transient or unstable. The protocol consisted of lysing cells to obtain crude chromatin (Crude Chr) and soluble fractions (Sol). A portion of the crude chromatin fraction was briefly treated with micrococcal nuclease to release polynucleosomes, which were collected with an ultracentrifugation step (Chr). Fractions tested were: whole cell lysate (W), soluble fraction (S), crude chromatin (Crude Chr), high-speed supernatant (Hs) and Chromatin (Chr). The cells were untreated or treated for 90 min with 0.1-M hydroxyurea to induce DNA damage prior to lysate preparation. Anti-Sir2 recognizes two specific bands not observed in a sir2Δ strain, the more rapidly migrating of which is likely to be an N-terminal proteolytic product, since the antiserum was raised to the 13 C-terminal amino acids of Sir2 (not shown). (C) Myc-tagged esa1–414 was recruited 0.2 kb downstream of the DSB at 2 and 3 h after break induction. ChIP anti-Myc of a control untagged strain (white) and of an esa1–414–13MYC tagged strain transformed with vector (light gray) or overexpressing LYS20 (dark gray). The enrichment of Myc-tagged esa1–414 relative to input and to the control locus SCR1 is shown. Time points in all ChIP experiments were time 0 (no HO induction), 1 h of growth in galactose (induction of HO), 2 h in galactose and 3 h in galactose. (D) Lys20 overexpression promoted recruitment 0.2 kb downstream of the break. ChIP anti-Lys20 in the same strains as (C), with control (white) representing a no-antibody control. The enrichment of Lys20 is shown relative to input and to SCR1. (E) Esa1 was enriched 0.6 kb downstream of the DSB 1 and 2 h after break induction. Esa1 no longer bound after repair had started. ChIP anti-Myc in an esa1–414–13MYC tagged strain that can repair by HR because the chromosomal silent mating-type loci are present. The control (white) was an untagged lys20Δ lys21Δ strain, whereas light gray and dark gray columns corresponded to the Myc-tagged strain transformed with vector or with 2μ LYS20, respectively. The enrichment relative to input and to the control locus SMC2 was graphed. Time points tested were as in (C), except with the last time point as 1 h of growth in glucose medium, to repress HO and allow repair by HR. (F) Endogenous Lys20 was recruited to the DSB, however higher levels were recruited when the protein was overexpressed. ChIP anti-Lys20 in the same strains as (E). The control (white) is a lys20Δ lys21Δ strain. The vector strain had endogenous levels of Lys20 expression, whereas the LYS20 sample overexpressed LYS20. Data represent triplicate samples for three independent experiments. Error bars represent the standard deviation (SD). The student's t-test was used to assess statistical significance and was represented with asterisks as follows: *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 4.

The esa1–414 cells had defective histone acetylation and eviction at the DSB. (A) Histone H4K5 acetylation was defective in esa1–414 cells compared to wild-type. ChIP anti-H4K5Ac in wild-type and esa1–414 strains. (B) Histone eviction at the break was impaired in esa1–414 cells. ChIP anti-H3Ct in strains in (A). (C) Relative H4 acetylation to histone levels increased after break induction in wild-type but not in esa1–414 cells. (D) Localized H4K5 acetylation increased when lys20-cat was overexpressed in esa1 cells 1 and 2 h after break induction. ChIP anti-H4K5Ac in esa1–414 lys20Δ lys21Δ strains transformed with vector (white), lys20-cat (light gray) or lys20-moon (dark gray). Time points and normalization are as in Figure 3E. (E) The histone eviction defect in esa1–414 cells was rescued by lys20-cat overexpression. Histone H3 levels were tested in the same strains as in (D) with anti-H3Ct ChIPs before, during and after HO break induction. (F) H4K5 acetylation relative to histone levels dramatically increased when lys20-cat was overexpressed. The enrichment of H4K5 acetylation at the break is shown relative to histone levels. Because histone H4 and H3 are heterodimers in DNA (52), comparison of histone H4 acetylation relative to histone H3 reflects histone/nucleosome occupancy. (G) Histone H4K16 acetylation increased equally in esa1 strains transformed with lys20-cat and lys20-moon. Histone H4K16 acetylation was tested in the same strains as in (D). (H) Relative histone H4K16 acetylation only increased when lys20-cat was overexpressed. H4K16 acetylation is shown relative to histone levels. (I) The moonlighting domain of Lys20 was important for recruitment to the DSBs. ChIP anti-Lys20 in a lys20Δ lys21Δ strain expressing lys20-cat or lys20-moon. Data represent three independent experiments. SD and P-values are the same as in Figure 3.

Figure 5.

LYS20 overexpression enhanced INO80 recruitment in esa1 cells. (A) Suppression of esa1 by Lys20 overexpression was dependent on the INO80 complex. Double mutants combining esa1–414 with mutants impairing different remodeling complexes were tested for suppression by LYS20 overexpression. Single mutant controls are indicated on the right. Deletion of catalytic subunits of INO80 and RSC is lethal (32,57). The ino80Δ strain is viable in the S288c background, but not in W303, which is used here (27). The INO80 complex (assayed using arp8Δ) proved necessary for suppression by Lys20 as increased dosage of Lys20 antagonized repair when the INO80 complex was disrupted, a result also observed in the single ARP8 null. (B) Ino80 recruitment to the break was similar in wild-type and esa1 cells. ChIP anti-Myc in INO80–9MYC and esa1–414 INO80–9MYC strains. (C) Overexpression of lys20-cat promoted increased recruitment of Ino80, 1 and 2 h after break induction in esa1 cells. ChIP anti-Myc in esa1–414 INO80–9MYC lys20Δ lys21Δ strains transformed with vector, lys20-cat and lys20-moon. Normalization, SD and P-values are the same as in Figure 3. (D) Ino80-myc and Lys20 interacted physically. When Ino80 is precipitated with an anti-Myc antibody, lys20-cat and lys20-moon co-immunoprecipitate. Input (in) and immunoprecipitated (IP) fractions are shown. The first two lanes in each blot were prepared from a strain expressing untagged Ino80 and overexpressing lys20-cat. Lanes 3–6 were from a strain expressing Ino80-myc and lys20-cat or lys20-moon from 2-μ plasmids. Note that compared to whole cell lysates in (E), IP samples of both myc-tagged Ino80 and Lys20 are proteolytically processed. (E) Ino80 protein levels were unaffected by Lys20 overexpression. Whole cell lysates of strains in (C) were probed with anti-Myc. (F) Histone H2A phosphorylation upon DNA damage was normal in esa1 strains transformed with vector or overexpressing LYS20. Protein lysates of the indicated strains were probed with anti-phospho-H2A and anti-H2A antibodies after being treated with hydroxyurea (0.2 M for 90 min). Histone expression is reduced when cells are treated with hydroxyurea (64), thus the damage-induced increase in H2A phosphorylation is only apparent when the H2A phosphorylation signal is quantified relative to H2A levels. Approximately 2-fold induction was observed for all strains tested. The quantification was performed for two independent samples.

Figure 6.

LYS20 overexpression suppressed esa1 histone eviction defect at the breaks. Cartoon representing a simplified chromatin landscape in wild-type, esa1 and esa1+LYS20 strains. The impaired histone acetylation (red balls) and histone eviction at the DSBs in esa1 cells were rescued by LYS20 overexpression through increased recruitment of INO80 (blue circles). Histone H2A phosphorylation (yellow pentagons) was unaffected.