A genetic and structural study of genome rearrangements mediated by high copy repeat Ty1 elements

Abstract

Ty elements are high copy number, dispersed repeated sequences in the Saccharomyces cerevisiae genome known to mediate gross chromosomal rearrangements (GCRs). Here we found that introduction of Ty912, a previously identified Ty1 element, onto the non-essential terminal region of the left arm of chromosome V led to a 380-fold increase in the rate of accumulating GCRs in a wild-type strain. A survey of 48 different mutations identified those that either increased or decreased the rate of Ty-mediated GCRs and demonstrated that suppression of Ty-mediated GCRs differs from that of both low copy repeat sequence- and single copy sequence-mediated GCRs. The majority of the Ty912-mediated GCRs observed were monocentric nonreciprocal translocations mediated by RAD52-dependent homologous recombination (HR) between Ty912 and a Ty element on another chromosome arm. The remaining Ty912-mediated GCRs appeared to involve Ty912-mediated formation of unstable dicentric translocation chromosomes that were resolved by one or more Ty-mediated breakage-fusion-bridge cycles. Overall, the results demonstrate that the Ty912-mediated GCR assay is an excellent model for understanding mechanisms and pathways that suppress genome rearrangements mediated by high copy number repeat sequences, as well as the mechanisms by which such rearrangements occur.

Figure 1. Assay model and a summary of the aCGH data.

A. Schematic of the −Ty and +Ty912 GCR assays on their respective chromosome Vs. Genes and Ty912 are not drawn to scale. B. Example of the Ty912-TEL05L deletion. Absolute intensities of the probes in the deletion region are noticeably lower than other regions where DNA is present. Signal spikes in the deletion represent redundant sequences (telomeric sequence on the left and DSF1 and HXT13 sequences in the middle). C. Example of a Class II duplication. The log2 ratio of intensities indicates a doubling of the genomic content from YLRWTy1-3 to TEL12R. D. Overview of the aCGH data. Filled squares represent telomeres, filled circles centromeres. Solid triangles represent full-length Ty1 elements. Hollow triangles represent solo deltas. The orientations of the triangles reflect the transcriptional orientation of the elements. Filled diamonds represent loci containing multiple Ty1 elements transcriptionally oriented in opposite directions. Lines above the chromosome arms represent duplicated regions. Orange lines represent duplications of genomic DNA between a telomere and a telomere oriented Ty element. Green lines represent duplications of genomic DNA between a telomere and a centromere oriented Ty element. Black lines represent duplications oriented between a telomere and a mixed orientation multiple Ty loci site. Ty1 elements targeted by independent translocations 3 or more times include YCRWdelta10, YCRWdelta11, YDRWTy1-5, YERWdelta21, YJRWTy1-2, YLRWTy1-3, YLRCdelta21, the YLRCdelta9/YLRWTy1-2/YLRCdelta12 multiple Ty loci, YNLCTy1-1, and YPRWTy1-3.

Figure 2. Ty912 mediates GCRs.

A. Southern blot of isolates I8–I17 using a probe specific to MCM3, an essential gene on chromosome V. The MCM3 probe was amplified using JCP447 & JCP 448. A PFGE reference column is included. B. An analysis of sequenced fusion Tys mediating the translocations. SNPs are attributable to either Ty912 (black), the target Ty(s) bordering the duplication (gold or magenta), or neither (blue). For isolate I17, the analysis was performed in three steps; SNPs consistent with YJRWTy1-1 were first colored gold, then SNPs consistent with YJRWTy1-2 were colored magenta, and finally SNPs belonging to either Ty912 or none of the Tys above were colored black and blue, respectively.

Figure 3. Analysis of isolate I9 reveals a structure consistent with breakage-fusion-bridge.

A. Deletion of the region between TEL05L and Ty912 in isolate I9. B. Duplication of the genomic region between TEL13L and YMLWTy1-1A/B. The duplicated region is located upstream of the 5′ end of YMLWTy1-1A/B as indicated. C. Schematic of the integration of pRDK1564 near the fusion junction and the subsequent steps utilized to create a vector cloning the fusion junction. The linearized form of the vector measures approximately 30 kb; 25 kb of the cloned fusion vector is predicted to be Ty DNA. D. Internal primer hybridizing within the epsilon sequence of the Ty reveals a heterogeneous chromatogram, indicating presence of at least two unique Ty sequences. E. A proposed mechanism for the creation of the observed structure. Exposed Ty912 sequence invades YMLWTy1-1A, resulting in a dicentric chromosome. Formation of the dicentric results in one round of breakage-fusion-bridge (with the break at the vertical arrow) followed by a subsequent break due to the formation of a second dicentric. The new break invades a wild type copy of chromosome XIII at YMLWTy1-1A/B resulting in the observed duplication and correctly sized fusion junction.

Figure 4. The dicentric structure of isolate I14 is resolved by Ty-mediated recombination.

A. Deletion of the region between TEL05L and Ty912 in isolate I14. B. Duplication of the region between YERCdelta14 and TEL05R. C. Schematic of the integration of pRDK1564 near the fusion junction and subsequent steps utilized to create a vector cloning the fusion junction. The Ty fusion junction measures approximately 6 kb, the size of one full length Ty1 element. D. Exposed Ty912 DNA invades YERCTy1-1, copying through the centromere and creating a dicentric chromosome. The unstable dicentric chromosome breaks at YERCdelta14 and invades YERWdelta20b, copying the rest of the duplicated region to TEL05R. * is a verified junction; † a predicted junction.

Figure 5. I12's tripartite recombination involves breakage-fusion-bridge.

A. Deletion of the region between TEL05L and Ty912 in isolate I12. B. Duplication of the region between TEL13L and YMLWTy1-1A/B. Triplication of the region between YMLWTy1-1A/B and YMLWTy1-2. C. Schematic of the integration of pRDK1564 near the fusion junction on chromosome V and subsequent steps utilized to create a vector cloning the fusion junction. The Ty fusion vector measures approximately 12 kb, as would be expected if Ty912 recombined with YMLWTy1-1A. D. Map of Southern probes and sizes of the expected fragments. DNA from the wild-type −Ty (WT −Ty), wild-type +Ty912 (WT +Ty912), and I12 were cut with restriction enzyme Acl I and subsequently probed with the indicated Southern probes. Bands within a dotted box represent equivalently sized bands. E. Schematic of the integration of pRDK1564 near a copy of YMLWTy1-2 and subsequent steps utilized to create a vector cloning the fusion junction. Results indicated two copies of the triplicated region fused together in an inverted fashion with the Ty fusion measuring approximately 11.4 kb, as would be expected if YMLWTy1-2 underwent a break-fusion-bridge cycle. F. Exposed Ty912 DNA invades YMLWTy1-1A on chromosome XIII. This leads to the formation of a dicentric that then leads to a breakage fusion bridge event at the vertical arrow followed by another break; the broken chromosome is repaired by mediating a translocation to homologous DNA on the wild type chromosome XIII. * indicates verified junctions.