An Assay to Study Intra-Chromosomal Deletions in Yeast

Abstract

An accurate DNA damage response pathway is critical for the repair of DNA double-strand breaks. Repair may occur by homologous recombination, of which many different sub-pathways have been identified. Some recombination pathways are conservative, meaning that the chromosome sequences are preserved, and others are non-conservative, leading to some alteration of the DNA sequence. We describe an in vivo genetic assay to study non-conservative intra-chromosomal deletions at regions of non-tandem direct repeats in Schizosaccharomyces pombe. This assay can be used to study both spontaneous breaks arising during DNA replication and induced double-strand breaks created with the S. cerevisiae homothallic endonuclease (HO). The preliminary genetic validation of this assay shows that spontaneous breaks require rad52⁺ but not rad51⁺, while induced breaks require both genes, in agreement with previous studies. This assay will be useful in the field of DNA damage repair for studying mechanisms of intra-chromosomal deletions.

Keywords: DNA double-strand breaks; Genetic Recombination; Yeast.

Figure 1

An assay to study spontaneous and induced double-strand breaks at regions of non-tandem repeats. (A). The ura-his-ura assay. In this assay, two non-functional ura4 alleles flank a functional his3⁺ allele. The ura4 alleles have 200 bps of identical overlapping sequences, creating two non-tandem repeats (gray areas). The S. cerevisiae homothallic endonuclease (HO) is cloned just upstream of the his3⁺ gene. The HO enzyme is on a LEU2 plasmid under the control of the nmt1 promoter which can be repressed with thiamine. Spontaneous ura4⁺his3⁻ recombinants are assayed by growing cells in EMM+UraHisAdeLeu media for 48 h then plating on selective EMM-Uracil. Induced break recombinants are assayed by growing cells for 48 h in media without thiamine to de-repress the HO endonuclease, while maintaining selection for the plasmid (EMM-Leucine). Cells are then plated on EMM-Uracil. All experiments were performed at 32 °C. (B). Box plot showing the frequency of recombinants for both induced and spontaneous breaks. (C). PCR across the ura-his-ura cassette in both pre- and post-recombination strains. Half arrowheads in (A) show approximate positions of primers.

Figure 2

Genetic requirements for spontaneous breaks. Box plots showing the spontaneous recombination frequency per 10⁵ colonies. Cells were grown on EMM-Uracil plates for 3–5 days at 32 °C. For clarity, insets are shown for strains with similar recombination frequencies.

Figure 3

Genetic requirements for induced breaks. Box plots showing the HO-endonuclease-induced recombination frequency per 10⁴ colonies. Cells were grown on EMM-Uracil plates for 3–5 days at 32 °C. For clarity, insets are shown for strains with similar recombination frequencies.