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. 2022 Jun 24;50(12):6870–6889. doi: 10.1093/nar/gkac520

Figure 1.

Figure 1.

BIR promotes deletions between microhomologies flanking inverted DNA repeats. (A) The lys2-InsH reporter before (0 h) and after (7 h) DSB. Top. BIR is initiated by induction of a DSB by HO endonuclease at MATa located in a truncated (recipient) chromosome (R) of yeast disomic for Chr. III. Invasion of one broken end of the recipient into the homologous full-length donor (D) chromosome containing MATα-inc establishes BIR synthesis that can progress approximately 100kb to the end of the chromosome. The lys2-InsH (Lys−) reporter consists of 69bp inverted repeats (yellow) separated by a 9bp spacer (purple) and is flanked by 9bp direct repeats (red). The construct was inserted at three different positions along the track of BIR synthesis in the donor chromosome: “MAT” (at MATα-inc), “16kb” centromere distal from MATα-inc, and “36 kb” centromere distal from MATα-inc. Bottom. Replication slippage during BIR proceeding through the lys2-InsH reporter can result in deletion of InsH producing a functional allele (LYS2*) conferring a Lys+ phenotype. Positions of primers (FP: forward primer, RP: reverse primer) for confirming deletions by PCR are shown for both LYS2* and lys2-InsH alleles. (B) Lys+ reversion rates for the strains containing lys2-InsH reporter cassette at the three positions specified in (A) prior to DSB induction (DSB 0hr), following BIR (DSB 7 h), as well as in isogenic strains lacking the HO cut-site (No DSB). Asterisks indicate statistically significant difference (P < 0.005) from Lys+ reversion rates at 0 h (DSB 0 h). Numbers above bars indicate median rates (see Supplementary Data S2 for precise P-values and 95% CIs). (C) Representative gel image showing PCR-amplified products of the lys2-InsH allele and the LYS2* allele (404 bp) present in a single BIR outcome (lane 1). PCR products of WT LYS2 (374 bp) (lane 3) and the lys2-InsH construct before BIR (530 bp) (lane 4) are also shown for comparison, along with a 100-bp DNA ladder (L) in lane 2. Primers used for all PCR products are those shown in (A, bottom) (FP and RP). Analysis of lys2-InsH deletion products (like LYS2*) was performed by Sanger sequencing.