The Yku70-Yku80 complex contributes to regulate double-strand break processing and checkpoint activation during the cell cycle

Abstract

DNA double-strand breaks (DSBs) are repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). HR requires 5' DSB end degradation that occurs in the presence of cyclin-dependent kinase (CDK) activity. Here, we show that a lack of any of the NHEJ proteins Yku (Yku70-Yku80), Lif1 or DNA ligase IV (Dnl4) increases 5' DSB end degradation in G1 phase, with ykuDelta cells showing the strongest effect. This increase depends on MRX, the recruitment of which at DSBs is enhanced in ykuDelta G1 cells. DSB processing in G2 is not influenced by the absence of Yku, but it is delayed by Yku overproduction, which also decreases MRX loading on DSBs. Moreover, DSB resection in ykuDelta cells occurs independently of CDK activity, suggesting that it might be promoted by CDK-dependent inhibition of Yku.

Figure 1

The absence of non-homologous end joining allows double-strand break resection in G1. YEP+raf α-factor-arrested cell cultures of wild-type JKM139 and yku80Δ, yku70Δ yku80Δ, dnl4Δ and lif1Δ derivative strains were transferred to α-factor-containing YEP+raf+gal to induce HO expression (time zero). (A) Fluorescence-activated cell sorting analysis of DNA content. (B) Schematic representation of the system used to detect DSB resection. Gel blots of SspI-digested genomic DNA separated on alkaline agarose gel were hybridized with a single-stranded RNA probe specific for the unresected strand at the MAT locus, which shows HO-cut and uncut fragments of 0.9 and 1.1 kb, respectively. 5′-to-3′ resection progressively eliminates SspI sites located 1.7, 3.5, 4.7, 5.9, 6.5, 8.9 and 15.8 kb centromere-distal from the HO-cut site, producing larger SspI fragments (r1–r7) detected by the probe. (C) Analysis of ssDNA formation as described in (B). (D) Densitometric analysis of the representative experiment shown in (C). Three independent experiments were performed with similar results. (E) Western blot analysis of protein extracts with Rad53 antibodies. DSB, double-strand break; ssDNA, single-stranded DNA; wt, wild type; YEP, yeast extract peptone.

Figure 2

Yku prevents Mre11-dependent double-strand break processing and efficient Mre11 recruitment to double-strand breaks in G1. (A–F) YEP+raf α-factor-arrested cell cultures of wild-type JKM139 and yku80Δ, yku80Δ mre11Δ, yku80Δ mre11-H125N, MRE11-MYC and yku70Δ yku80Δ MRE11-MYC derivative strains were transferred to α-factor-containing YEP+raf+gal (time zero). (A) Fluorescence-activated cell sorting analysis of DNA content. (B) Analysis of ssDNA formation as described in Fig 1B. (C) Densitometric analysis of the representative experiment shown in (B). Three independent experiments were performed with similar results. (D) Western blot analysis of protein extracts with Rad53 antibodies. (E) Representative ChIP time-course analysis of Mre11–DSB association. Quantitative PCRs before (Input) and after Mre11-Myc immunoprecipitation (Mre11-IP). Twofold serial dilutions of the input DNA establish the linear range of PCR. The efficiency of Mre11-Myc immunoprecipitation was similar at all time points (data not shown). (F) Quantitative analysis of Mre11–DSB association. Densitometric data from four independent experiments as in (E) were expressed as the relative fold enrichment of DSB over control (CON) signal independently for each time point and normalized to input DNA samples background. Error bars indicate s.d. (G,H) YEP+raf α-factor-arrested cell cultures of JKM139 MRE11-MYC carrying either the empty vector or the 2μ URA3 EXO1 plasmid were transferred to YEP+raf+gal to induce HO expression (time zero). (G) Representative ChIP time-course analysis of Mre11–DSB association. Quantitative PCRs before (Input) and after Mre11 immunoprecipitation (Mre11-IP) with anti-Myc antibodies. (H) Quantitative analysis of Mre11–DSB association. Densitometric data from three independent experiments as in (G) were expressed as described in (F). Error bars indicate s.d. ChIP, chromatin immunoprecipitation; DSB, double-strand break; Exp, exponentially growing cells; ssDNA, single-stranded DNA; wt, wild type; YEP, yeast extract peptone.

Figure 3

The absence of Yku does not influence double-strand break processing and Mre11 recruitment at double-strand breaks in G2. YEP+raf nocodazole-arrested cell cultures of wild-type JKM139 and isogenic yku70Δ yku80Δ cells, expressing the MRE11-MYC allele, were transferred to nocodazole-containing YEP+raf+gal (time zero). (A) DSB resection analysis was performed as described in Fig 1B. (B) Densitometric analysis of the representative experiment shown in (A). Four independent experiments were performed with similar results. (C) Representative ChIP time-course analysis of Mre11–DSB association as described in Fig 2E. (D) Quantitative analysis of Mre11–DSB association. Densitometric data from three independent experiments as in (C) were expressed as in Fig 2F. Error bars indicate s.d. ChIP, chromatin immunoprecipitation; CON, control; DSB, double-strand break; wt, wild type; YEP, yeast extract peptone.

Figure 4

Yku overproduction impairs 5′-end processing and Mre11 loading at an HO-induced double-strand break. (A–E) YEP+raf nocodazole-arrested cell cultures of JKM139 strains carrying either empty vectors or both the 2μ URA3 YKU70 and 2μ LEU2 YKU80 plasmids, all expressing the MRE11-MYC allele, were transferred to nocodazole-containing YEP+raf+gal (time zero). (A) DSB resection analysis as described in Fig 1B. (B) Densitometric analysis of the representative experiment shown in (A). Three independent experiments were performed with similar results. (C) Representative ChIP time-course analysis performed as described in Fig 2E. (D) Quantitative analysis of Mre11–DSB association. Densitometric data from three independent experiments as in (C) were expressed as in Fig 2F. Error bars indicate s.d. (E) Western blot analysis of protein extracts with Rad53 antibodies. (F) Exponentially growing YEP+raf cell cultures of mec1Δ LSY1259 (10 Ty1-HOcs-HIS3) strains transformed with 2μ URA3 plasmids, either empty or carrying both YKU70 and YKU80, were transferred to YEP+raf+gal to induce HO expression (time zero). Western blot analysis of protein extracts with Rad53 antibodies is shown. ChIP, chromatin immunoprecipitation; CON, control; DSB, double-strand break; Exp, exponentially growing cells; YEP, yeast extract peptone.

Figure 5

Interaction between Yku and cyclin-dependent kinase in the regulation of double-strand break resection. (A–C) YEP+raf nocodazole-arrested cell cultures of wild-type JKM139 and yku80Δ, GAL-SIC1ΔNT and GAL-SIC1ΔNT yku80Δ derivative strains were transferred to nocodazole-containing YEP+raf+gal (time zero). (A) Analysis of DSB resection as described in Fig 1B. (B) Densitometric analysis of the representative experiment shown in (A). Three independent experiments were performed with similar results. (C) Western blot analysis of protein extracts with Rad53 antibodies. (D,E) YEP+raf α-factor-arrested cell cultures of wild-type JKM139 and GAL-CLB2 or yku80Δ derivative strains were transferred to α-factor-containing YEP+raf+gal (time zero). (D) Analysis of DSB resection as described in Fig 1B. (E) Densitometric analysis of the representative experiment shown in (D). Three independent experiments were performed with similar results. (F,G) ChIP analysis of Yku70 binding to an HO-induced DSB. (F) YEP+raf α-factor- (G1) or nocodazole-arrested (G2) cell cultures of a JKM139 derivative strain expressing the YKU70-MYC allele were transferred to α-factor- or nocodazole-containing YEP+raf+gal, respectively (time zero). (G) YEP+raf α-factor-arrested cell cultures of wild-type and GAL-CLB2 isogenic strains expressing the YKU70-MYC allele were transferred to α-factor-containing YEP+raf+gal (time zero). In both (F) and (G), densitometric data from three independent experiments were expressed as in Fig 2F. Error bars indicate s.d. ChIP, chromatin immunoprecipitation; DSB, double-strand break; Exp, exponentially growing cells; wt, wild type; YEP, yeast extract peptone.