Telomeric amplicons of SUL1 and Y' in yeast are generated by microhomology-mediated break induced replication occurring in cis

Abstract

Gene amplification is a potent driver of evolution and is thought to contribute to genetic diseases, including cancer. The yeast Saccharomyces cerevisiae is a powerful organism for understanding amplification mechanisms. When yeast is grown long term in sulfate-limiting chemostats, amplification of the gene that encodes the primary sulfate transporter, SUL1, is a common outcome. Here we describe a form of SUL1 amplification in which multiple copies of the right terminal region of chromosome II are appended in tandem to a native telomere. We find this form of amplicon when we delete the origin of replication next to SUL1 or delete a variety of genes involved in DNA metabolism. It is the only form of amplification found in a yku70{Delta} mutant suggesting that unprotected telomeres are involved. We propose that these terminal addition events occur when the unprotected 3 G1-3T telomeric sequence invades a short ([~]7 bp) internal telomere sequence (ITS) to begin a form of microhomology-mediated break-induced replication (mmBIR) that has been documented in type-I survivors of telomerase mutants. In addition to amplification of the right end of chromosome II we also find that telomeres containing the sub-telomeric repeat Y experience similar tandem amplification events and show that their formation is reduced in a pol32{Delta} mutant, a gene required for mmBIR. Within individual amplicons the ITSs and Ys are nearly identical, suggesting that the multiple copies of the amplified region are generated in a single mmBIR event that we describe as pseudo-rolling circle mmBIR. A similar amplification event at the P-telomere of human chromosome 18 has four copies of a [~]54 kb region separated by ITSs of nearly identical size. This finding suggests that these additional copies of the terminal fragment of human chromosome 18 arose by the same pseudo-rolling circle mechanism, perhaps during a period of telomeric stress. AUTHOR SUMMARYThe human genome is peppered with duplicates (or higher numbers) of segments that are located at sites both nearby and distant from the original, ancestral segments. These Copy Number Variants, or CNVs, appear to be highly variable among different individuals and are being examined with great interest as potential loci associated with genetic disease. Experimentally determining how these CNVs arise and become distributed across the genome is nearly impossible using humans. We are using budding yeast as the model organism to explore mechanisms of gene amplification. In this work we show that by destabilizing the ends of yeast chromosomes (telomeres) or by interfering with genes involved in the replication, repair, or recombination of DNA results in a specific form of segmental copy number increase that is initiated at telomeres. We propose that a telomere invades an internal chromosome site and sets up a pseudo-circular template for conservative DNA replication. The outcome is a chromosome with multiple, identical copies of a chromosome end arranged in tandem. We believe that it is also a major mechanism used by cells to repair telomeres that have become eroded during aging.