GSK-3β Homolog Rim11 and the Histone Deacetylase Complex Ume6-Sin3-Rpd3 Are Involved in Replication Stress Response Caused by Defects in Dna2

Abstract

Lagging strand synthesis is mechanistically far more complicated than leading strand synthesis because it involves multistep processes and requires considerably more enzymes and protein factors. Due to this complexity, multiple fail-safe factors are required to ensure successful replication of the lagging strand DNA. We attempted to identify novel factors that are required in the absence of the helicase activity of Dna2, an essential enzyme in Okazaki-fragment maturation. In this article, we identified Rim11, a GSK-3β-kinase homolog, as a multicopy suppressor of dna2 helicase-dead mutant (dna2-K1080E). Subsequent epistasis analysis revealed that Ume6 (a DNA binding protein, a downstream substrate of Rim11) also acted as a multicopy suppressor of the dna2 allele. We found that the interaction of Ume6 with the conserved histone deacetylase complex Sin3-Rpd3 and the catalytic activity of Rpd3 were indispensable for the observed suppression of the dna2 mutant. Moreover, multicopy suppression by Rim11/Ume6 requires the presence of sister-chromatid recombination mediated by Rad52/Rad59 proteins, but not vice versa. Interestingly, the overexpression of Rim11 or Ume6 also suppressed the MMS sensitivity of rad59Δ. We also showed that the lethality of dna2 helicase-dead mutant was attributed to checkpoint activation and that decreased levels of deoxynucleotide triphosphates (dNTPs) by overexpressing Sml1 (an inhibitor of ribonucleotide reductase) rescued the dna2 mutant. We also present evidence that indicates Rim11/Ume6 works independently but in parallel with that of checkpoint inhibition, dNTP regulation, and sister-chromatid recombination. In conclusion, our results establish Rim11, Ume6, the histone deacetylase complex Sin3-Rpd3 and Sml1 as new factors important in the events of faulty lagging strand synthesis.

Keywords: Dna2; GSK-3b; HDAC; checkpoint; genome instability.

Figure 1

Rim11 and its substrate Ume6 are multicopy suppressors of dna2-KE. (A) Overexpression of Rim11 suppressed dna2-KE and it is dependent on its kinase activity. Briefly, YJA18JK65 (dna2Δ::HIS3, pRS316-Dna2, and pRS314-Dna2-K1080E) was transformed with pRS315-Dna2 (positive control), vector only (none, negative control), pRS325-Rim11, or pRS325-Rim11-KA (K68A) under the control of each native promoter. Transformants were inoculated in SD liquid medium (3 ml) and grown until saturation. Each saturated culture was spotted in 10-fold serial dilutions on SD plates without (control) or with 5-FOA (0.1%) followed by incubation for 4–5 days at 30°. (B) Expression of Rim11 and Rim11-KA proteins were examined by Western blot using anti-FLAG antibody. Nonspecific band (∼100 kDa) that always appears with anti-FLAG antibody was used as internal loading control. (C) Overexpression of Ume6, not Ime1, suppressed dna2-KE mutant. (D) Expression of Ime1 from a multicopy plasmid was confirmed by Western blot using anti-FLAG antibody, while a nonspecific band serves as a loading control. (E) Ume6 is working downstream of Rim11 in the rescue of dna2-KE mutant defect. (F) Zn²⁺-dependent DNA binding activity of Ume6 is indispensable for the suppression of dna2-KE. (G) Ume6-6C6A protein level was examined by Western blotting using anti-FLAG antibody.

Figure 2

A role for Ume6 in DNA replication and repair. (A) Cells lacking Ume6 are impeded in S-phase progression. (B) Ume6 protein level diminished when exposed to DNA damaging agent, MMS. Logarithmically growing cells with endogenously tagged (myc) Ume6 were grown in YPD liquid media with or without the presence of 0.033% MMS for 1.5 hr. Total proteins were extracted and subjected to Western blot analysis. (C) Ume6 is required in cells resistance against DNA damage. Cells (200–300 cells) of the indicated strains were spread onto YPD media with or without the indicated concentrations of CPT, HU, MMS, and 4-NQO. Plates were grown at 30° for ∼3–6 days and the number of surviving colonies were counted. Data are representative of three independent experiments. WT, wild type.

Figure 3

Rim11/Ume6 suppressed the lethality of dna2-KE mutant in a manner dependent on the histone deacetylase complex, Sin3-Rpd3. (A) Ume6 interaction with Sin3 is essential and not its transcription regulation activity per se. (B) The expression level of Ume6 mutants were examined by Western blot using anti-FLAG antibody. (C) Ume6-dependent suppression of dna2-KE requires the presence of both Sin3 and Rpd3. dna2-KE mutant devoid of either SIN3 or RPD3 was transformed with 2µ-based plasmid expressing either Rim11 or Ume6. (D) Overexpression of Sin3-Rpd3 did not rescue dna2-KE. (E) Rpd3 deacetylase activity is essential for the Ume6-dependent suppression of dna2-KE. (F) The expression levels of Rpd3 wild-type and deacetylase-negative proteins were examined by Western blot.

Figure 4

Ume6 is working independently of Rad52/Rad59. (A) Multicopy suppression by Rim11/Ume6 requires the presence of Rad52 and Rad59. The Rad52/Rad59-dependent suppression of dna2-KE does not require (B) Rim11, Ume6, (C) Sin3, or Rpd3. (D) Ume6 and Rad52 showed synergistic interaction in the suppression of dna2-KE. Data are representative of three independent experiments.

Figure 5

RIM11 and UME6 genetically interact with homologous recombination genes. (A) Overexpression of Rim11 or Ume6 suppressed the MMS sensitivity of rad59Δ. Briefly, rad51Δ, rad52Δ, and rad59Δ cells were transformed with plasmids as indicated in the figure. Transformants were inoculated in SD medium and incubated at 30° until saturation. Serial dilutions (10-fold) of the indicated transformants were spotted onto SD medium with or without the indicated concentrations of MMS and incubated at 30° for 5–6 days. (B) Double mutants rad51Δ ume6Δ, rad52Δ ume6Δ, and rad59Δ ume6Δ showed increased sensitivity to MMS and HU. Cells (200–300 cells) of the indicated strains were spread onto YPD media with or without the indicated concentrations of MMS and HU. Plates were grown at 30° for ∼3–6 days and the number of surviving colonies were counted. Data are representative of three independent experiments. (C) A summary of the genetic interactions observed between RIM11/UME6 and the recombination genes, RAD51, RAD52, and RAD59. The Rim11/Ume6 pathway appears to compensate specifically for the Rad59-dependent role of Rad52 (bold font). WT, wild type.

Figure 6

Inviability of dna2-KE is caused by checkpoint activation. (A) Inviability of dna2-KE is suppressed by second site mutations: rad9Δ, rad53-R605A, and rad53Δ sml1Δ. (B) Ume6 mode of suppression is independent of that of rad9Δ. Data are representative of three independent experiments.

Figure 7

Reduced levels of dNTP is beneficial for dna2-KE cells. (A) Sml1 is a multicopy suppressor of dna2-KE defects and acts in parallel with Rad52/Rad59, Rim11/Ume6, and Yen1 pathways. (B) Sml1 and Ume6 showed synergistic interaction in the suppression of dna2-KE. Data are representative of three independent experiments.

Figure 8

The copy number of dna2-KE mutant allele influenced the severity of cell growth defects. (A) A single copy of dna2-KE produced viable cells. (B) Introduction of multiple copies of dna2-KE allele caused cells inviability. WT, wild type.

Figure 9

Multiple pathways to alleviate damages caused by helicase-deficient Dna2. (A) Sister-chromatid, recombination-mediated suppression of dna2 mutant requires the strand-annealing activity of Rad52 and its interaction with Rad59, together with cohesion factors Elg1 and Rsc2. (B) The nuclease activity of Rad27 cleaves DNA flaps, compensating the need for Dna2. (C) Defect in the helicase activity of Dna2 gives rise to toxic recombination intermediates that can be resolved by Yen1 in anaphase. Disruption of G₂/M checkpoint by deletion of RAD9 allows unrestrained entry of dna2 cells into anaphase. (D) Rim11-dependent phosphorylation of Ume6 may protect Ume6 against degradation in the presence of DNA damage. The DNA binding activity of Ume6 provides targeted recruitment of the histone deacetylase complex, Sin3-Rpd3. (E) Downregulation of checkpoint activation may (i) permit dna2-KE cells to escape G₂/M arrest and proceed to anaphase where Yen1 is activated, (ii) allow unrestrained recruitment of Rad52 to sites of damage, and (iii) prevent the destruction of Sml1 which controls the levels of dNTP. Types of suppression: * indicates Multicopy suppression; ** indicates Synthetic rescue; *** indicates Synthetic sickness.