FLPrecombinase induction of the breakage-fusion-bridge cycle and gene conversion inSaccharomyces cerevisiae

✍ Scribed by G. H. Rank; W. Xiao; A. Kolenovsky; G. Arndt

• Publisher: Springer-Verlag
• Year: 1988
• Tongue: English
• Weight: 859 KB
• Volume: 13
• Category: Article
• ISSN: 0172-8083
• DOI: 10.1007/bf00424420

✦ Synopsis

A YEp chimaeric plasmid containing URA3 and SMR1 [sulfometuron methyl resistant (SM R) allele of IL V2] as selectable markers, and the 2 #m site-specific recombination FLP recognition target (FRT), was integrated at the ilv2-A1 site in chromosome XIlI in a [cir °] haploid. Southern analysis defined two integrant structures. Structure I had URA3 distal and SMR1 proximal to FRT whereas in structure II both markers were distal to FRT. Selectable markers were stably inherited in [cir °] haploids and [cO" °] diploids heterozygous for the integrant and IL V2. Approximately 14% of heterozygous [cir +] diploid cells exhibited homozygotization for the distal (500 kb) ade4 marker in trans. In [cir + ] diploids FLP-FRT recombination resulted in the simultaneous loss of both structure II markers, whereas the structure I distal URA3 marker loss always preceded the variable loss of the proximal SMR1 marker. URAcells continued to segregate for loss of SMR1 until stable URA-SM g or URA-SM s cells were produced. Gene conversion was identified in stable URA-SM R cells that were homozygous SMR1/SMR1 but contained wild type ILV2 restriction endonuclease sites. These observations support a model based on concerted FLP-FRT action resulting from the secondary integration of native 2 gm DNA followed by unequal sister chromatid exchange (USCE) within inverted FRTs. The resultant chromatid bridge resulted in a double-stand break. Fusion of the broken ends of sister chromatids generated a breakage-fusion-bridge cycle (BFBC). Repeated rounds of the BFBC resulted in proximal marker loss and the generation of additional double-strand breaks. Recombinogenic properties of the double-strand break initiated events leading to homozygotization and gene conversion.