Distinct roles of Mus81, Yen1, Slx1-Slx4, and Rad1 nucleases in the repair of replication-born double-strand breaks by sister chromatid exchange

Abstract

Most spontaneous DNA double-strand breaks (DSBs) arise during replication and are repaired by homologous recombination (HR) with the sister chromatid. Many proteins participate in HR, but it is often difficult to determine their in vivo functions due to the existence of alternative pathways. Here we take advantage of an in vivo assay to assess repair of a specific replication-born DSB by sister chromatid recombination (SCR). We analyzed the functional relevance of four structure-selective endonucleases (SSEs), Yen1, Mus81-Mms4, Slx1-Slx4, and Rad1, on SCR in Saccharomyces cerevisiae. Physical and genetic analyses showed that ablation of any of these SSEs leads to a specific SCR decrease that is not observed in general HR. Our work suggests that Yen1, Mus81-Mms4, Slx4, and Rad1, but not Slx1, function independently in the cleavage of intercrossed DNA structures to reconstitute broken replication forks via HR with the sister chromatid. These unique effects, which have not been detected in other studies unless double mutant combinations were used, indicate the formation of distinct alternatives for the repair of replication-born DSBs that require specific SSEs.

Fig 1

Molecular analysis of the effects of mus81 and yen1 mutations in SCE. (A) Schemes of plasmid pRS316-TINV and the intermediates produced by SCE after HO cleavage. Sizes of the XhoI-SpeI bands detected with the LEU2 probe (line with asterisks) are indicated. SCR intermediates physically detected correspond to an unstable dicentric plasmid that it is not recovered as a final product in Leu⁺ recombinant colonies. (B) Kinetics and quantification of DSBs and SCE intermediates after different times of HO induction in galactose in the following strains: wild type (WT; WSR-7D), mus81Δ (WSR-M81), yen1Δ (WSR-Y1), and mus81Δ yen1Δ (WSR-M81Y1) and the catalytic mutant mus81-dd (WSR-M81DD). A representative Southern analysis is shown for each genotype analyzed. Quantification of DSBs (1.4-kb and 2.4-kb bands) and SCE (4.7-kb band) was calculated relative to the total DNA of each lane. Averages and standard deviations (bars) of at least three independent experiments are shown for each time point and genotype. (C) Effects of mus81Δ and yen1Δ in spontaneous recombination (-HO) and DSB-induced SCE (+HO) frequencies, as determined with Leu⁺ recombinants, using the inverted repeat system TINV after 5 h of HO activation with 2% galactose, working with the same strains as in panel B. Each value represents the average of three median values obtained from three different fluctuation tests, each performed with 6 independent colonies from three different transformants for each genotype. Asterisks indicate statistically significant differences compared to wild type according to Student's t test (P < 0.001).

Fig 2

Genetic and physical analyses of the effects of MUS81-MMS4 overexpression in SCR in yen1Δ strains. (A) Physical analysis of the effects of MUS81-MMS4 overexpression in DSB-induced SCE. Kinetics of DSBs and SCE intermediates in isogenic wild-type (WSR-7D), mus81Δ (WSR-M81), and yen1Δ (WSR-Y1) strains transformed with pWDH800 carrying the active heterodimer MUS81-MMS4 or the catalytically inactive heterodimeric mus81-dd-MMS4 under the control of the GAL1,10 promoter. (B) Effects of MUS81-MMS4 overexpression on spontaneous recombination (-HO) and DSB-induced SCE (+HO) frequencies in the TINV inverted repeat system. Wild-type (WT) and mutant strains were transformed with empty vector pRS314 (-) or with pWDH800 carrying either active heterodimeric MUS81-MMS4 or catalytically inactive heterodimeric mus81-dd-MMS4 under the control of the GAL1,10 promoter. Asterisks indicate statistically significant differences between the strains carrying either the active MUS81-MMS4 or the catalytically inactive mus81-dd-MMS4 and the strains with the empty vector, according to Student's t test (*, P < 0.001; **, P < 0.005). Other details for the experiment were those described for Fig. 1.

Fig 3

Genetic and physical analyses of the effects of YEN1 overexpression in SCR in mus81Δ strains. (A) Physical analysis of the effects of Yen1 overexpression in SCE. Wild-type (WT) and mus81Δ strains were transformed with empty vector pAG414GPD-ccdB-HA (-) or the vector carrying either the active YEN1 or the catalytically inactive yen1^E193A/E195A (yen1-ee) allele. Averages and standard deviations (bars) of at least three independent experiments are shown for each time point and genotype. (B) Genetic analysis of the effects of Yen1 overexpression on spontaneous recombination (-HO) and DSB-induced SCE (+HO) frequencies in the TINV inverted repeat system. Wild-type, mus81Δ, and yen1Δ strains were transformed with empty vector pAG414GPD-ccdB-HA (-) or vector carrying either the active YEN1 or the catalytically inactive yen1^E193A/E195A (yen1-ee) allele under the control of the GPD1 promoter. Asterisks indicate statistically significant differences between the strains carrying either the active YEN1 or the catalytically inactive yen1^E193A/E195A (yen1-ee) and the strains with the empty vector, according to Student's t test (*, P < 0.001; **, P < 0.005). Other details for the experiment were those described for Fig. 1.

Fig 4

Effects of slx4Δ in SCR. (A) Physical analysis of the effects of slx4Δ in different genetic backgrounds in DSB-induced SCR. Kinetics and quantification of DSBs and DSB-induced SCE intermediates in isogenic wild-type (WT; WSR-7D), slx4Δ (WSR-S4), slx4Δ mus81Δ (WSR-S4M81), slx4Δ yen1Δ (WSR-S4Y1), and slx4Δ mus81Δ yen1Δ (WSR-S4M81Y1) strains are shown. (B) Genetic analysis of the effects of slx4Δ on spontaneous recombination (-HO) and DSB-induced SCE (+HO) frequencies in the TINV inverted repeat system. Asterisks indicate statistically significant differences compared to wild type according to Student's t test (*, P < 0.001; **, P < 0.005). Other details for the experiment were those described for Fig. 1.

Fig 5

Analysis of genetic interactions between MUS81, YEN1, and SLX4. Genetic analysis of the effects of overexpression of Mus81-Mms4 and Yen1 in slx4Δ strains with or without mus81Δ or yen1Δ on spontaneous recombination (-HO) and DSB-induced SCE (+HO) frequencies in the TINV inverted repeat system are shown. WT, wild type. Asterisks indicate statistically significant differences between the strains expressing either active MUS81-MMS4 or active YEN1 and the strains with the empty vector, according to Student's t test (*, P < 0.001; **, P < 0.005). Other details for the experiment were those described for Fig. 1.

Fig 6

Physical analysis of SCR in rad1Δ and slx1Δ cells. Physical analysis of DSB formation and SCR in various mutants: (A) slx4Δ (WSR-S4), rad1Δ (WSR-R1), and slx4Δ rad1Δ (WSR-S4R1); (B) slx1Δ (WSR-S1), rad1Δ (WSR-R1), and slx1Δ rad1Δ (WSR-S1R1); (C) mus81Δ. WT, wild type. Other details for the experiment were those described for Fig. 1.

Fig 7

Genetic analysis of the effects of rad1Δ and slx1Δ on Leu⁺ recombinants generated by the TINV system. Frequencies of spontaneous and HO-induced Leu⁺ recombinants are shown for slx1Δ and rad1Δ mutants, as well as double, triple, and quadruple mutant combinations with mus81Δ and yen1Δ. WT, wild type. Other details for the experiment were those described for Fig. 1.

Fig 8

Mus81-Mms4 and Yen1 define two different resolution pathways for the repair of replication-born DSBs by SCR. A nick can lead to a DSB during replication regardless of whether it occurs in the leading or lagging strand (the lagging strand is shown here). Repair by strand invasion and DNA synthesis with the sister chromatid lead to the formation of a D-loop and/or HJ, which would result in SCR following incision by either Mus81-Mms4 (epistatic to Slx4 in this scenario) or Yen1. Rad1 would likely be required to cleave the single-strand tail generated by the heterologous short HO site after strand invasion in our assay, but a role in junction cleavage cannot be discarded. RF, replication fork.