The role of Holliday junction resolvases in the repair of spontaneous and induced DNA damage

Abstract

DNA double-strand breaks (DSBs) and other lesions occur frequently during cell growth and in meiosis. These are often repaired by homologous recombination (HR). HR may result in the formation of DNA structures called Holliday junctions (HJs), which need to be resolved to allow chromosome segregation. Whereas HJs are present in most HR events in meiosis, it has been proposed that in vegetative cells most HR events occur through intermediates lacking HJs. A recent screen in yeast has shown HJ resolution activity for a protein called Yen1, in addition to the previously known Mus81/Mms4 complex. Yeast strains deleted for both YEN1 and MMS4 show a reduction in growth rate, and are very sensitive to DNA-damaging agents. In addition, we investigate the genetic interaction of yen1 and mms4 with mutants defective in different repair pathways. We find that in the absence of Yen1 and Mms4 deletion of RAD1 or RAD52 have no further effect, whereas additional sensitivity is seen if RAD51 is deleted. Finally, we show that yeast cells are unable to carry out meiosis in the absence of both resolvases. Our results show that both Yen1 and Mms4/Mus81 play important (although not identical) roles during vegetative growth and in meiosis.

Figure 1.

Schematic representation of DSB repair by homologous recombination and its products. Following the formation of a DSB there is single-strand resection to form a 3′ overhang, which invades a homologous sequence. Single end invasion can be resolved through: (A) SDSA—strand disengagement, ligation to form a NCO product, or (B) The D-loop can be nicked and ligation may lead to a CO product. When there is also a second end capture by the D-loop, polymerization can lead to the formation of a double HJ, which can either be (C) resolved by HJ resolvases to be ligated to form NCO and CO products, or (D) undergo dissolution by the activity of a helicase and a topoisomerase to form a NCO product.

Figure 2.

yen1Δ mms4Δ cells show growth defects. (A) Tetrad analysis of a double heterozygous diploid. Colony size indicates that yen1Δ mms4Δ colonies have a slow growth phenotype. yen1Δ single deletion (circle), mms4Δ single deletion (square) and yen1Δ mms4Δ double deletion (diamond) are marked. (B) Calculated doubling time of wild-type cells compared to single (yen1Δ and mms4Δ) and double mutant yen1Δ mms4Δ. Quantification of growth rate shows a prolonged doubling time for the double deletion yen1Δ mms4Δ.

Figure 3.

Genetic interactions of yen1Δ and mms4Δ with different repair enzymes. (A) Drop assay to analyze MMS sensitivity of yen1Δ, mms4Δ and double deletion yen1Δ mms4Δ with repair enzymes (rad18Δ, rad52Δ, rad1Δ or rad51Δ). (B) Tetrad analysis of double deletion yen1Δ mms4Δ combined with mutations in additional repair enzymes. The yen1Δ mms4Δ strain (diamond) and the triple deletion (square) are marked. (C) Doubling time of yen1Δ, mms4Δ and yen1Δ mms4Δ combined with mutations in additional repair enzymes.

Figure 4.

Role of resolvases in the repair of DSB. (A) Schematic representation of the system used to examine DSB repair of a single-induced DSB. The endogenous URA3 gene on Ch. V carries an HO endonuclease recognition site. In the LYS2 locus on Ch. II, there is an insertion of 5.6 kb sequence homologous to the URA3 sequence, with a mutated HOcs (HOcs-inc) and two polymorphisms of BamHI (B) and EcoRI (R) sites. The cells also contain the HO endonuclease under a galactose-inducible GAL1 promoter. Following the transfer of the cells to galactose, a single DSB is formed on Ch. V. The repair of the break can lead to either non-crossover or crossover products. Restriction enzyme sites (arrowhead) and fragment sizes expected in a Southern blot are indicated. (B) Graphic representation of the repair efficiency of wild-type, yen1Δ, mms4Δ and yen1Δ mms4Δ cells. Repair efficiency is calculated by comparing the number of cells grown on glucose compared to colonies formed on galactose-containing medium. (C) Southern blot of DNA from cells taken 0, 2, 10 or 24 h after transfer to galactose-containing medium. The DNA was digested with PvuII and ApaLI and probed with a fragment of Ch. V carrying the URA3 gene. The percentage of crossover product was calculated in the 24 h time point by densitometer quantification of the autoradiogram. The percentage of the crossover bands was divided by the total DNA content (Ch. II, Ch. V and cross-over). The numbers in parentheses are the standard deviation (SD) values for three independent experiments.

Figure 5.

Meiotic defects in the absence of Yen1 and Mms4. Graphic representation of the percentage of sporulating cells in wild-type, double heterozygous yen1Δ mms4Δ, homozygous yen1Δ, homozygous mms4Δ and double homozygous yen1Δ mms4Δ. Diploid cells were incubated at 25°C for 5 days in sporulation medium and were analyzed under the microscope for the presence of meiotic spores. For each strain, three independent samples were examined. The wild-type strain, the double heterozygous and the homozygous yen1Δ strain show high sporulation (∼50%); the homozygous mms4Δ strain shows reduced sporulation (16%) and almost no spores (0.072%) were observed in the double homozygous yen1Δmms4Δ strain.