Ku prevents Exo1 and Sgs1-dependent resection of DNA ends in the absence of a functional MRX complex or Sae2

Abstract

In this study, we investigate the interplay between Ku, a central non-homologous end-joining component, and the Mre11-Rad50-Xrs2 (MRX) complex and Sae2, end-processing factors crucial for initiating 5'-3' resection of double-strand break (DSB) ends. We show that in the absence of end protection by Ku, the requirement for the MRX complex is bypassed and resection is executed by Exo1. In contrast, both the Exo1 and Sgs1 resection pathways contribute to DSB processing in the absence of Ku and Sae2 or when the MRX complex is intact, but functionally compromised by elimination of the Mre11 nuclease activity. The ionizing radiation sensitivity of a mutant defective for extensive resection (exo1Δ sgs1Δ) cannot be suppressed by the yku70Δ mutation, indicating that Ku suppression is specific to the initiation of resection. We provide evidence that replication-associated DSBs need to be processed by Sae2 for repair by homologous recombination unless Ku is absent. Finally, we show that the presence of Ku exacerbates DNA end-processing defects established in the sae2Δ sgs1Δ mutant, leading to its lethality.

Figure 1

Suppression of the mre11Δ IR sensitivity by YKU70 deletion. Exponentially growing cells of the indicated genotypes were 1:10 serially diluted, spotted onto YPD or selective plates and exposed to the indicated IR dose.

Figure 2

Phenotype of mre11-nd and mre11-nd sgs1Δ mutants. (A) Suppression of the mre11-nd IR sensitivity by the yku70Δ mutation (quantitation in (B)) or high-copy expression of EXO1 (quantitation in (C), ^*P=0.03, unpaired t-test). (D) Radiation sensitivity of mre11-nd mutants in conjunction with sgs1Δ or exo1Δ mutations. (E) Schematic representation of the chromosome III MAT locus used in the physical assay to assess resection of an HO-induced DSB. The 5′-3′ degradation destroys the StyI (S) and XbaI (X) recognition sites, which translates into the disappearance of the StyI/XbaI digestion fragments. (F) Southern blot analysis and (G) cut fragment intensity plots showing the kinetics of the cut fragment intensity disappearance as a ratio of the intensity 30 min after induction. The means from four experiments are presented, error bars indicate s.d.

Figure 3

Suppression of the sae2Δ mutant phenotype by YKU70 deletion. Radiation sensitivity of sae2Δ mutants: (A) spot assays and (B) survival plots as described in Figure 2C. ^*P=0.01 (unpaired t-test). (C) Epistatic relationship between sae2Δ and mre11-nd mutants, as shown by IR spot assays. Resection physical assay: (D) Southern blot analysis and (E) cut fragment intensity plots as described in Figure 2G. (F) Radiation sensitivity of sgs1Δ exo1Δ mutants, as indicated by spot assays. (G) CPT sensitivity of mre11-nd and sae2Δ mutants. Exponentially growing cells in SC minimal medium were 1:10 serially diluted and spotted on SC plates containing the indicated concentration of camptothecin in DMSO.

Figure 4

YKU70/YKU80 over-expression sensitizes sae2Δ and mre11-nd mutants to IR. (A) Spot assays and (B) survival plots of wild type, sae2Δ and mre11-nd mutants transformed with empty or YKU70/YKU80 over-expressing vectors. Exponentially growing cells in SC-Ura to maintain selection of the plasmids were 1:10 serially diluted, spotted onto SC-Ura plates and exposed to IR. The means from at least three experiments are presented, error bars indicate s.d.; ^*P=0.01, ^**P=0.009.

Figure 5

Genetic interactions between rad27Δ, mre11-nd, sae2Δ and sgs1Δ mutants. Viability and genotypes of spores derived from diploids heterozygous for the indicated mutations. (A) Deletion of YKU70 suppresses the synthetic lethality/growth defect of rad27Δ mutants with sae2Δ and mre11-nd, but not with rad55Δ. (B) Loss of Yku70 or EXO1 over-expression rescues the sae2Δ sgs1Δ synthetic lethality.

Figure 6

Telomere-associated phenotypes of sae2Δ sgs1Δ yku70Δ mutants. (A) Schematic representation of the telomeric Y′ elements and TG repeats. XhoI liberates a wide band of ∼1.3 kb in wild-type cells, which is used to evaluate telomere repeat length in other genetic backgrounds. (B) Southern blot analysis of XhoI-digested genomic DNA as detected with a Y′ probe. (C) Telomere length analysis after induction of YKU70 expression with galactose in the yku70Δ and sae2Δ sgs1Δ yku70Δ mutant. (D) Viability and genotypes of spores derived from diploids heterozygous for the indicated mutations.

Figure 7

Models for the interplay between resection machinery and Ku at DSB ends. In wild-type cells, DSBs can exist in three dynamic states: (A) Ku-bound, which blocks access to Exo1, (B) MRX-bound, which can initiate end processing and (C) MRX-Ku-bound, which can initiate NHEJ and removal of Ku is required to allow resection initiation. Recruitment of Sae2 in G2 and clipping of the ends allows access to the processive resection machinery and creates an intermediate that can no longer convert states and commit to HR. In nuclease-defective mutants of Mre11, though compromised for initial processing, the presence of Sae2 channels ends to HR and redundant activity from Sgs1-Dna2 allows initiation of resection. In the absence of SAE2, the MRX-bound ends can still initiate resection, presumably with some assistance by Sgs1, whereas the Ku-bound and MRX-Ku-bound ends are blocked. When the end protection by Ku is lost, in mre11-nd yku70Δ and sae2Δ yku70Δ for example, MRX-naked ends can be resected by Exo1. For the MRX-bound ends even if compromised for the initial clipping, the absence of Ku allows Sgs1 (and maybe Exo1) to assist in initiating resection of the DSB. Finally, in the absence of Mre11, where the only state present is the Ku-bound state, access of Exo1 is blocked in a Ku-dependent manner.